Medical devices for delivery of siRNA

ABSTRACT

Disclosed are medical devices that incorporate siRNA. In addition to one or more siRNA constructs, devices include nanostructures fabricated on a surface to form a nanotopography. A random or non-random pattern of structures may be fabricated such as a complex pattern including structures of differing sizes and/or shapes. Microneedles may be incorporated on devices. The pattern including nanostructures may be formed on the surface of the microneedles.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/328,723 having a filing date of Apr. 28, 2010, and to U.S.Provisional Patent Application Ser. No. 61/416,057 having a filing dateof Nov. 22, 2010, both of which are incorporated herein in theirentirety by reference.

BACKGROUND

Many factors affect gene expression in organisms. For instance, smallRNAs, generally 20 to 25 nucleotides in length, have emerged asimportant regulators of eukaryotic gene expression. One class of smallRNA is the short interfering RNA (siRNA). siRNA plays a role in the RNAinterference (RNAi) pathway, and particularly in RNA silencing, asequence-specific RNA degradation process that is triggered bydouble-stranded RNA (dsRNA). siRNAs are double-stranded with small 3′overhangs and derive from longer dsRNA precursors that induce silencing.They serve as guides to direct destruction of target RNA and have beenimplicated as primers in the amplification of dsRNA via the activity ofa cellular RNA dependent RNA polymerase.

Since their discovery, synthetic siRNA have been produced that mayinduce RNAi in mammalian cells through silencing or otherwisesuppressing transcription of multiple genes. However, despite promisingresults, problems still exist with successful utilization of siRNAtechnology, among which delivery methods play a large role. Typically,siRNA have been delivered either by direct injection, electroproration,or by complexing with a transfecting agent. However, the siRNA remainsactively present for only a matter of hours following delivery. In orderto obtain longer lasting effectiveness, improved delivery methods mustbe found that may provide steady, long-term delivery of siRNA.

Transdermal delivery devices that provide a route for siRNA to bedelivered in an active state at effective, steady concentrations over aperiod of time would be of great benefit. Many difficulties must beovercome to reach this goal. For instance, the human body has developedmany systems to prevent the influx of foreign substances such asenzymatic degradation in the gastrointestinal tract, structuralcomponents that prevent absorption across epithelium, hepatic clearance,and immune and foreign body response.

Transdermal devices have been developed for sustained delivery ofcertain drugs including those for treatment of vertigo, contraception,and smoking addiction. In order to be successful, a transdermal devicemust deliver an agent across the epidermis, which has evolved with aprimary function of keeping foreign substances out. The outermost layerof the epidermis, the stratum corneum, has structural stability providedby overlapping corneocytes and crosslinked keratin fibers held togetherby coreodesmosomes and embedded within a lipid matrix, all of whichprovide an excellent barrier function. Beneath the stratum corneum isthe stratum granulosum, within which tight junctions are formed betweenkeratinocytes. Tight junctions are barrier structures that include anetwork of transmembrane proteins embedded in adjacent plasma membranes(e.g., claudins, occludin, and junctional adhesion molecules) as well asmultiple plaque proteins (e.g., ZO-1, ZO-2, ZO-3, cingulin, symplekin).Tight junctions are found in internal epithelium (e.g., the intestinalepithelium, the blood-brain barrier) as well as in the stratumgranulosum of the skin. Beneath both the stratum corneum and the stratumgranulosum lays the stratum spinosum. The stratum spinosum includesLangerhans cells, which are dendritic cells that may become fullyfunctioning antigen-presenting cells and may institute an immuneresponse and/or a foreign body response to an invading agent.

Unfortunately, transdermal delivery methods are presently limited todelivery of low molecular weight agents that have a moderatelipophilicity and no charge. Even upon successful crossing of thenatural boundary, problems still exist with regard to maintaining theactivity level of delivered agents and avoidance of foreign body andimmune response.

The utilization of supplementary methods to facilitate transdermaldelivery of active agents has improved this delivery route. Forinstance, microneedle devices have been found to be useful in transportof material into or across the skin. In general, a microneedle deviceincludes an array of needles that may penetrate the stratum corneum ofthe skin and reach an underlying layer. Examples of microneedle deviceshave been described in U.S. Pat. No. 6,334,856 to Allen, et al. and U.S.Pat. No. 7,226,439 to Prausnitz, et al., both of which are incorporatedherein by reference.

While the above describes improvement in the art, room for furtherimprovement exists.

SUMMARY

In accordance with one embodiment of the present invention, a device fordelivery of an siRNA construct across a dermal barrier. The devicecomprises a microneedle and a plurality of nanostructures fabricated ona surface of the microneedle, the nanostructures being arranged in apredetermined pattern. An siRNA construct is in fluid communication withthe microneedle.

In accordance with another embodiment of the present invention, a methodfor delivering an siRNA construct across a dermal barrier. The methodcomprises penetrating the stratum corneum with a microneedle. Themicroneedle comprises a plurality of nanostructures formed on a surfaceof the microneedle and arranged in a pattern. The siRNA construct is influid communication with the microneedle, the siRNA construct beingtransported across the stratum corneum following penetration of thestratum corneum by the microneedle.

In accordance with yet another embodiment of the present invention, amethod for forming a device for delivery of an siRNA construct across adermal barrier is disclosed. The method comprises fabricating an arrayof microneedles; fabricating a pattern of nanostructures on a surface ofat least one of the microneedles; and associating an siRNA constructwith the microneedles such that the siRNA construct is in fluidcommunication with the microneedles.

BRIEF DESCRIPTION OF THE DRAWINGS

A full and enabling disclosure of the subject matter, including the bestmode thereof, directed to one of ordinary skill in the art, is set forthmore particularly in the remainder of the specification, which makesreference to the appended figures in which:

FIG. 1 illustrates one embodiment of a microneedle device.

FIG. 2 illustrates another embodiment of a microneedle device.

FIG. 3 illustrates one embodiment of a microneedle including a surfacethat defines a nanotopography that may interact with an extracellularmatrix (ECM).

FIG. 4 illustrates one embodiment of a complex pattern that may beformed on a microneedle surface.

FIG. 5 illustrates a pattern including multiple iterations of thecomplex pattern of FIG. 4.

FIG. 6 illustrates a Sierpinski triangle fractal.

FIGS. 7A-7D illustrate complex fractal and fractal-likenanotopographies.

FIG. 8 illustrates another complex pattern that may be formed on amicroneedle surface.

FIG. 9 illustrates exemplary packing densities as may be utilized fornano-sized structures as described herein including a square packingdesign (FIG. 9A), a hexagonal packing design (FIG. 9B), and a circlepacking design (FIG. 9C).

FIGS. 10A-10C schematically illustrate a nanoimprinting method as may beutilized in one embodiment in forming a device.

FIG. 11 schematically illustrates one embodiment of a device.

FIG. 12 is a perspective view of one embodiment of a transdermal patchprior to delivery of a drug compound.

FIG. 13 is a front view of the patch of FIG. 12.

FIG. 14 is a perspective view of the patch of FIG. 12 in which therelease member is partially withdrawn from the patch.

FIG. 15 is a front view of the patch of FIG. 12.

FIG. 16 is a perspective view of the transdermal patch of FIG. 122 afterremoval of the release member and during use.

FIG. 17 is a front view of the patch of FIG. 16.

FIG. 18 is a perspective view of another embodiment of a transdermalpatch prior to delivery of a drug compound.

FIG. 19 is a front view of the patch of FIG. 18.

FIG. 20 is a perspective view of the patch of FIG. 19 in which therelease member is partially peeled away from the patch.

FIG. 21 is a front view of the patch of FIG. 20.

FIG. 22 is a perspective view of the patch of FIG. 18 in which therelease member is completely peeled away from the patch.

FIG. 23 is a perspective view of the transdermal patch of FIG. 18 afterremoval of the release member and during use.

FIGS. 24A-24E illustrate several nanotopography patterns as describedherein.

FIG. 25 is an SEM of a film including a nanopatterned surface.

FIGS. 26A and 26B are two SEM of a film including another nanopatternedsurface.

FIG. 27 is an SEM of a film including another nanopatterned surface.

FIG. 28 is an SEM of a film including another nanopatterned surface.

FIG. 29 is an SEM of a film including another nanopatterned surface.

FIG. 30 is an SEM of a film including another nanopatterned surface.

FIG. 31 is an SEM of a film including another nanopatterned surface.

FIG. 32 is an SEM of a film including another nanopatterned surface.

FIG. 33 is an SEM of a film including another nanopatterned surface.

FIG. 34 graphically illustrates the effects on permeability to bovineserum albumin (BSA) in a monolayer of cells on polystyrene filmspatterned with nanopatterns as described herein.

FIG. 35 graphically illustrates the effects on permeability toimmunoglobulin-G (IgG) in a monolayer of cells on polystyrene filmspatterned with nanopatterns as described herein.

FIGS. 36A and 36B are 3D live/dead flourescein staining images showingparacellular transport of IgG across a monolayer of cells on apolystyrene patterned surface as described herein.

FIG. 37 graphically illustrates the effects on permeability to BSA in amonolayer of cells on polypropylene films patterned with nanopatterns asdescribed herein.

FIG. 38 graphically illustrates the effects on permeability to IgG in amonolayer of cells on polypropylene films patterned with nanopatterns asdescribed herein.

FIGS. 39A and 39B are 3D live/dead flourescein staining images showingparacellular transport of IgG across a monolayer of cells on apolypropylene patterned surface as described herein.

FIGS. 40A-40F are scanning electron microscopy (SEM) images of cellscultured on nanopatterned surfaces as described herein.

FIG. 41 is an array of microneedles including a surface layer defining apattern of nanostructures thereon.

FIG. 42 is a single microneedle of the array of FIG. 41.

FIG. 43 graphically illustrates the effects on permeability to siRNA ina monolayer of cells on a polypropylene film patterned as describedherein.

DETAILED DESCRIPTION OF REPRESENTATIVE EMBODIMENTS

Reference now will be made in detail to various embodiments of thedisclosed subject matter, one or more examples of which are set forthbelow. Each example is provided by way of explanation, not limitation.In fact, it will be apparent to those skilled in the art that variousmodifications and variations may be made in the present disclosurewithout departing from the scope or spirit of the subject matter. Forinstance, features illustrated or described as part of one embodimentmay be used on another embodiment to yield a still further embodiment.Thus, it is intended that the present disclosure covers suchmodifications and variations as come within the scope of the appendedclaims and their equivalents.

In general, a device for delivery of siRNA constructs is disclosed. Morespecifically, the device may include a plurality of microneedles at asurface and a pattern of structures fabricated on the microneedles. Atleast a portion of the structures are fabricated on a nanometer scale.The device is also associated with one or more siRNA constructs, forinstance in a layer of the device or in a reservoir that is in fluidcommunication with the surface that includes the microneedles.

Without wishing to be limited to any particular theory, it is believedthat the pattern of nanostructures, i.e., the nanotopography, of adevice may improve delivery of siRNA while minimizing foreign body andimmune response. Through utilization of a device, siRNA may be targetedfor delivery to a specific site, e.g., a specific tissue or cell type ina specific delivery area, or may be delivered in a systemic fashion, forinstance via the cardiovascular system.

The siRNA agents of the devices are sufficiently short that they do nottrigger a deleterious non-specific interferon response in normalmammalian cells. Thus, the administration of a composition including oneor more siRNA agents may be used to affect transcription of a targetgene, while circumventing an interferon response as well as minimizingforeign body response. The siRNA of a device may generally include aduplexed region of less than 50, less than 40, or less than 30nucleotide pairs, for instance between about 20 and about 25 pairs. Ingeneral, an siRNA polynucleotide comprises a double-stranded RNA (dsRNA)but is not intended to be so limited and may comprise a single-strandedRNA.

An siRNA may be formed according to any known process. For instance, ansiRNA may be synthesized synthetically or via transcription of a DNAconstruct either in vivo or in vitro. In general, an siRNA molecule maybe synthesized using methods, reagents and equipment available to one ofskill in the art. By way of example, siRNA may be designed andengineered using computer software available commercially from variousvendors, e.g., OligoEngine (Seattle, Wash.); Dharmacon, Inc. (Lafayette,Colo.); Ambion Inc. (Austin, Tex.); and QIAGEN, Inc. (Valencia,Calif.)). See also Elbashir et al., 2000 Genes & Development 15:188-200;Elbashir et al., 2001 Nature 411:494-98.

According to such methods, a cDNA sequence may be scanned for targetsequences that have AA dinucleotides. Sense and anti-senseoligonucleotides may be generated to these targets that contain a G/Ccontent of, for example, about 35 to 55%. These sequences may then becompared to others in the human genome database to minimize homology toother known coding sequences (e.g. by performing a BLAST search usingthe information available through the NCBI database).

An siRNA polynucleotide molecule may be generated by in vitro or in vivotranscription of suitable DNA sequences (e.g., polynucleotide sequencesencoding a target polypeptide, or a desired portion thereof). The DNAmay be incorporated into a vector with a suitable RNA polymerasepromoter (such as for example, T7, U6, H1, or SP6 although otherpromoters may be equally useful). Endogenous RNA polymerases within acell may mediate transcription in vivo or cloned RNA polymerase may beused for transcription in vivo or in vitro. For transcription from atransgene or an expression construct, a regulatory region may be used totranscribe siRNA strands.

A vector may be delivered to a subject by, for example, intravenousinjection, local administration (U.S. Pat. No. 5,328,470 to Nabel, etal.), or by stereotactic injection (see, e.g., Chen et al., Proc. Natl.Acad. Sci. USA 91:3054-3057, 1994). A DNA template may include twotranscription units, one that produces a transcript that includes thesense strand of an siRNA agent and one that produces a transcript thatincludes the antisense strand of an siRNA agent. When the templates aretranscribed following delivery, an siRNA agent may be produced.

Polynucleotides that comprise an siRNA may be derived from asingle-stranded polynucleotide that comprises a single-strandedoligonucleotide fragment (e.g., of about 18-30 nucleotides) and itsreverse complement, typically separated by a spacer sequence. Cleavageof the spacer may provide a single-stranded oligonucleotide fragment andits reverse complement, and they may anneal to form, optionally withadditional processing steps that may result in addition or removal ofone, two, three or more nucleotides from the 3′ end and/or the 5′ end ofeither or both strands, a double-stranded siRNA polynucleotide. Thespacer may be of a length that permits the fragment and its reversecomplement to anneal and form a double-stranded structure (e.g., like ahairpin polynucleotide) prior to cleavage of the spacer, and optionally,subsequent processing steps that may result in addition or removal ofone, two, three, four, or more nucleotides from the 3′ end and/or the 5′end of either or both strands. A spacer sequence may therefore be anypolynucleotide sequence that is situated between two complementarypolynucleotide sequence regions which, when annealed into adouble-stranded nucleic acid, result in an siRNA polynucleotide.

A formed siRNA polynucleotide may have blunt ends. Optionally, at leastone strand of the siRNA polynucleotide may have one or more nucleotidesoverhanging at the 3′ end. For instance, each strand of an siRNApolynucleotide duplex may have a two-nucleotide overhang at the 3′ end.The two-nucleotide overhang may be a thymidine dinucleotide (TT) but mayalso comprise other bases, for example, a TC dinucleotide or a TGdinucleotide, or any other dinucleotide. The overhang dinucleotide mayalso be complementary to the two nucleotides at the 5′ end of thesequence of the polynucleotide that is targeted for interference. For adiscussion of 3′ ends of siRNA polynucleotides see, e.g., WO 01/75164,to Tuschl, et al., which is incorporated herein by reference. Adouble-stranded siRNA structure may be formed by a singleself-complementary RNA strand or two complementary RNA strands.

An siRNA polynucleotide may comprise other naturally occurring,recombinant, or synthetic single-stranded or double-stranded polymers ofnucleotides (ribonucleotides or deoxyribonucleotides or a combination ofboth) and/or nucleotide analogues (e.g., an oligonucleotide orpolynucleotide or the like, typically in 5′ to 3′ phosphodiesterlinkage).

Inhibition is sequence-specific in that nucleotide sequencescorresponding to the duplex region of the RNA are targeted for geneticinhibition. Accordingly, siRNA containing nucleotide sequences identicalto a portion of the target gene may be preferred for inhibition.However, siRNA sequences with insertions, deletions, and single pointmutations relative to the target sequence may also be effective forinhibition and are encompassed herein. For instance, an siRNA mayinclude modifications to either the phosphate-sugar backbone or thenucleoside.

An siRNA compound may exhibit variability by differing (e.g., bynucleotide substitution, including transition or transversion) at one,two, three or four nucleotides from a particular sequence. Thesedifferences may occur at any of the nucleotide positions of a particularsiRNA, depending on the length of the molecule, whether situated in asense or in an antisense strand of the double-stranded polynucleotide.The nucleotide difference may be found on one strand of adouble-stranded polynucleotide, where the complementary nucleotide withwhich the substitute nucleotide would typically form hydrogen bond basepairing may not necessarily be correspondingly substituted.

Sequence identity may be optimized by alignment algorithms known in theart and calculating the percent difference between the nucleotidesequences. Alternatively, the duplex region of the RNA may be definedfunctionally as a nucleotide sequence that is capable of hybridizingwith a portion of the target gene transcript.

Following formation, an siRNA polynucleotide may be tested for theability to interfere with the expression of the target polypeptideaccording to methods known in the art. The determination of theeffectiveness of an siRNA polynucleotide includes not only considerationof its ability to interfere with the expression of the targetpolypeptide, but also whether the siRNA polynucleotide is toxic to thehost cell. For example, a desirable siRNA would exhibit an RNAinterference activity and would also not exhibit an unwanted biologicalconsequence. An example of an unwanted biological consequence isapoptosis of a cell for which cell death is not a desired as a result ofthe introduction of the siRNA into the host cell.

The properties of an siRNA agent, including its pharmacologicalproperties, may be influenced and tailored, for example by theintroduction of ligands, e.g. tethered ligands, or vectors, e.g., viralvectors, to the siRNA agent. In addition, pharmacological properties ofan siRNA may be improved by incorporating a ligand in a formulation ofan siRNA when the siRNA agent has a tethered ligand.

A wide variety of ligands may be tethered to an siRNA agent or used asformulation conjugate or additive, e.g., to the carrier of aligand-conjugated monomer subunit. Examples are described below in thecontext of a ligand-conjugated monomer subunit but it should beunderstood that ligands may be coupled at other points to an siRNAagent.

Ligands may be coupled, covalently or non-covalently, either directly orindirectly via an intervening tether to the carrier. A ligand ortethered ligand may be present on a ligand-conjugated monomer when theligand-conjugated monomer is incorporated into the growing strand. Inone embodiment, a ligand may be incorporated into a “precursor”ligand-conjugated monomer subunit after a “precursor” ligand-conjugatedmonomer subunit has been incorporated into the growing strand. Forexample, a monomer having, e.g., an amino-terminated tether, e.g.,TAP-(CH₂)_(n)NH₂ may be incorporated into a growing sense or antisensestrand. In a subsequent operation, i.e., after incorporation of theprecursor monomer subunit into the strand, a ligand having anelectrophilic group, e.g., a pentafluorophenyl ester or aldehyde group,may subsequently be attached to the precursor ligand-conjugated monomerby coupling the electrophilic group of the ligand with the terminalnucleophilic group of the precursor ligand-conjugated monomer subunittether.

A ligand may alter the distribution, targeting or lifetime of an siRNAagent into which it is incorporated. For instance, a ligand may providean enhanced affinity for a selected target, e.g., molecule, cell or celltype, compartment, e.g., a cellular or organ compartment, tissue, organor region of the body. A ligand may improve transport, hybridization,and specificity properties and may also improve nuclease resistance ofthe resultant natural or modified oligoribonucleotide.

A ligand may act as a therapeutic modifier, e.g., for enhancing uptake;a diagnostic compound or reporter group e.g., for monitoringdistribution; a cross-linking agent; a nuclease-resistance conferringmoiety; and/or a natural or unusual nucleobase; among other uses.Non-limiting examples may include lipophilic molecules, lipids, lectins,steroids (e.g., uvaol, hecigenin, diosgenin), terpenes (e.g.,triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol derivatizedlithocholic acid), vitamins, carbohydrates (e.g., a dextran, pullulan,chitin, chitosan, synthetic (e.g., Oligo Lactate 15-mer) and natural(e.g., low and medium molecular weight) polymers, insulin, cyclodextrinor hyaluronic acid), proteins, protein binding agents, integrintargeting molecules, polycationics, peptides, polyamines, and peptidemimics. Other examples include folic acid or epithelial cell receptorligands, such as transferin.

A ligand may be a naturally occurring or recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid.Examples of polyamino acids include polylysine (PLL), poly L-asparticacid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic moieties, e.g., cationic lipid,cationic porphyrin, quaternary salt of a polyamine, or an alpha helicalpeptide.

A ligand may include a targeting group, e.g., a cell or tissue targetingagent, e.g., a thyrotropin, melanotropin, surfactant protein A, mucincarbohydrate, a glycosylated polyaminoacid, transferrin, bisphosphonate,polyglutamate, polyaspartate, or an Arg-Gly-Asp (RGD) peptide or RGDpeptide mimetic.

A ligand may be a protein, e.g., glycoproteins, lipoproteins, e.g. lowdensity lipoprotein (LDL), or albumins, e.g. human serum albumin (HSA),or peptides, e.g., molecules having a specific affinity for a co-ligand,or, ntibodies e.g., an antibody, that binds to a specified cell typesuch as a cancer cell, endothelial cell, or bone cell. A ligand may be ahormone or hormone receptor. They may also include non-peptidic species,such as cofactors, multivalent lactose, multivalent galactose,N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, ormultivalent fucose.

A ligand may be a substance, e.g., a drug, which may increase the uptakeof the siRNA agent into a cell, for example, by disrupting the cell'scytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. A ligand may be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine,myoservin, tetracyclin.

A ligand may be a lipid or lipid-based molecule that may bind a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand mayallow for distribution of the conjugate to a target tissue, e.g., livertissue, including parenchymal cells of the liver. Other molecules thatmay bind HSA may also be used as ligands. For example, neproxin oraspirin may be used. A lipid or lipid-based ligand may (a) increaseresistance to degradation of the conjugate, (b) increase targeting ortransport into a target cell or cell membrane, and/or (c) may be used toadjust binding to a serum protein, e.g., HSA. A lipid based ligand maybe used to modulate, e.g., control the binding of the conjugate to atarget tissue. For example, a lipid or lipid-based ligand that binds toHSA more strongly will be less likely to be targeted to the kidney andtherefore less likely to be cleared from the body.

Viral and non-viral vectors may be utilized as delivery vehicles indelivery of siRNA constructs as is known in the art, any of which may beincorporated herein. As used herein, the term “vector” refers to anucleic acid molecule capable of transporting another nucleic acid towhich it has been linked. One type of vector is a plasmid, which refersto a circular double stranded DNA loop into which additional nucleicacid segments may be ligated. Another type of vector is a viral vector,wherein additional nucleic acid segments may be ligated into the viralgenome. Certain vectors are capable of autonomous replication in a hostcell into which they are introduced (e.g., bacterial vectors having abacterial origin of replication and episomal mammalian vectors). Othervectors (e.g., non-episomal mammalian vectors) are integrated into thegenome of a host cell upon introduction into the host cell, and therebyare replicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to as recombinantexpression vectors, or more simply expression vectors. In general,expression vectors of utility may be in the form of plasmids. In thepresent specification, plasmid and vector may be used interchangeably asthe plasmid is the most commonly used form of vector. However, thedisclosure is intended to include such other forms of expressionvectors, such as viral vectors (e.g., replication defectiveretroviruses, lentiviruses, adenoviruses and adeno-associated viruses),which serve equivalent functions. In one embodiment, lentiviruses may beused to deliver one or more siRNA molecules to a cell, e.g., amacrophage, T cell, dendritic cell, or hematopoletic stem cell.

The various components of a construct may be operably linked to oneanother, according to known practices. Within a vector, “operablylinked” is intended to mean that a nucleotide sequence of interest islinked to regulatory sequence(s) in a manner which allows for expressionof the nucleotide sequence (e.g., in a target cell when the vector isintroduced into the target cell). The term “regulatory sequence” isintended to include promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel; Gene Expression Technology: Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Regulatorysequences include those which direct constitutive expression of anucleotide sequence in many types of host cell and those which directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector may dependon such factors as the choice of the target cell, the level ofexpression of siRNA desired, and the like.

An siRNA construct may be incorporated into a delivery vehicle, e.g., aliposome, or a nano- or microparticle. For example, an siRNA may beencapsulated in a liposome as described in Liposome Technology, Vol. II,Incorporation of Drugs, Proteins, and Genetic Material, CRC Press. AnsiRNA, depending upon its solubility, may be present both in the aqueouslayer and in the lipidic layer, or in what is generally termed aliposomic suspension. The hydrophobic layer, generally but notexclusively, may include phospholipids such as lecithin andsphingomyelin, steroids such as cholesterol, ionic surfactants such asdiacetylphosphate, stearylamine, or phosphatidic acid, and/or othermaterials of a hydrophobic nature.

Depending on the particular sequence utilized and the dose ofdouble-stranded siRNA material delivered, an siRNA may provide partialor complete loss of function for the target gene. A reduction or loss ofgene expression in at least 99% of targeted cells has been shown forgenes, e.g. U.S. Pat. No. 6,506,559 to Fire, et al. Lower doses ofdelivered material and longer times after administration of the selectedsiRNA may result in inhibition in a smaller fraction of cells.

A delivery device may be used to deliver siRNA to a gene for inhibition.For example, a gene that is essential for replication of a pathogen,transmission of a pathogen, or maintenance of an infection may beinhibited using a delivery device. As another example, genes of cells atrisk for infection by a pathogen or already infected cells may betargeted. The target gene may be a pathogen or host gene responsible forentry of a pathogen into its host, drug metabolism by the pathogen orhost, replication or integration of the pathogen's genome, establishmentor spread of an infection in the host, or assembly of the nextgeneration of pathogens. Methods of prophylaxis (i.e., prevention ordecreased risk of infection), as well as reduction in the frequency orseverity of symptoms associated with infection, are encompassed. Adevice may be used in combination with other treatment regimens,including virostatic and virotoxic agents, antibiotic agents, antifungalagents, anti-inflammatory agents, as well as combination therapies, andthe like.

A device may be used for inhibiting gene expression of cancer-relatedgenes. By way of example, an siRNA of a device may silence a gene of acancer, including solid tumors and leukemias, including: apudoma,choristoma, branchioma, malignant carcinoid syndrome, carcinoid heartdisease, carcinoma (e.g., Walker, basal cell, basosquamous,Brown-Pearce, ductal, Ehrlich tumor, in situ, Krebs 2, Merkel cell,mucinous, non-small cell lung, oat cell, papillary, scirrhous,bronchiolar, bronchogenic, squamous cell, and transitional cell),histiocytic disorders, leukemia (e.g., B cell, mixed cell, null cell, Tcell, T-cell chronic, HTLV-II-associated, lymphocytic acute, lymphocyticchronic, mast cell, and myeloid), histiocytosis malignant, Hodgkindisease, immunoproliferative small, non-Hodgkin lymphoma, plasmacytoma,reticuloendotheliosis, melanoma, chondroblastoma, chondroma,chondrosarcoma, fibroma, fibrosarcoma, giant cell tumors, histiocytoma,lipoma, liposarcoma, mesothelioma, myxoma, myxosarcoma, osteoma,osteosarcoma, Ewing sarcoma, synovioma, adenofibroma, adenolymphoma,carcinosarcoma, chordoma, cranio-pharyngioma, dysgerminoma, hamartoma,mesenchymoma, mesonephroma, myosarcoma, ameloblastoma, cementoma,odontoma, teratoma, thymoma, trophoblastic tumor, adenocarcinoma,adenoma, cholangioma, cholesteatoma, cylindroma, cystadenocarcinoma,cystadenoma, granulosa cell tumor, gynandroblastoma, hepatoma,hidradenoma, islet cell tumor, Leydig cell tumor, papilloma, Sertolicell tumor, theca cell tumor, leiomyoma, leiomyosarcoma, myoblastoma,myoma, myosarcoma, rhabdomyoma, rhabdomyosarcoma, ependymoma,ganglioneuroma, glioma, medulloblastoma, meningioma, neurilemmoma,neuroblastoma, neuroepithelioma, neurofibroma, neuroma, paraganglioma,paraganglioma nonchromaffin, angiokeratoma, angiolymphoid hyperplasiawith eosinophilia, angioma sclerosing, angiomatosis, glomangioma,hemangioendothelioma, hemangioma, hemangiopericytoma, hemangiosarcoma,lymphangioma, lymphangiomyoma, lymphangiosarcoma, pinealoma,carcinosarcoma, chondrosarcoma, cystosarcoma phyllodes, fibrosarcoma,hemangiosarcoma, leiomyosarcoma, leukosarcoma, liposarcoma,lymphangiosarcoma, myosarcoma, myxosarcoma, ovarian carcinoma,rhabdomyosarcoma, sarcoma (e.g., Ewing, experimental, Kaposi, and mastcell), neoplasms (e.g., bone, breast, digestive system, colorectal,liver, pancreatic, pituitary, testicular, orbital, head and neck,central nervous system, acoustic, pelvic, respiratory tract, andurogenital), neurofibromatosis, and cervical dysplasia, and fortreatment of other conditions in which cells have become immortalized ortransformed. A device may be used in combination with other treatmentmodalities, such as chemotherapy, cryotherapy, hyperthermia, radial-iontherapy, and the like.

A device is not limited to any type of target gene or nucleotidesequence. However, the following classes of possible target genes arelisted for illustrative purposes as target genes: developmental genes(e.g., adhesion molecules, cyclin kinase inhibitors, Wnt family members,Pax family members, Winged helix family members, Hox family members,cytokines/lymphokines and their receptors, growth/differentiationfactors and their receptors, neurotransmitters and their receptors);oncogenes (e.g., ABL1, BCL1, BCL2, BCL6, CBFA2, CBL, CSF1R, ERBA, ERBB,EBRB2, ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS, LCK, LYN,MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET, SRC, TALI, TCL3,and YES); tumor suppressor genes (e.g., APC, BRCA1, BRCA2, MADH4, MCC,NF1, NF2, RB1, TP53, and WT1); and enzymes (e.g., ACC synthases andoxidases, ACP desaturases and hydroxylases, ADP-glucose pyrophorylases,ATPases, alcohol dehydrogenases, amylases, amyloglucosidases, catalases,cellulases, chalcone synthases, chitinases, cyclooxygenases,decarboxylases, dextrinases, DNA and RNA polymerases, galactosidases,glucanases, glucose oxidases, granule-bound starch synthases, GTPases,helicases, hemicellulases, integrases, inulinases, invertases,isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes,nopaline synthases, octopine synthases, pectinesterases, peroxidases,phosphatases, phospholipases, phosphorylases, phytases, plant growthregulator synthases, polygalacturonases, proteinases and peptidases,pullanases, recombinases, reverse transcriptases, RUBISCOs,topoisomerases, and xylanases).

The device includes, in addition to the siRNA construct(s), microneedlesupon which have been fabricated a plurality of nano-sized structures. Asutilized herein, the term ‘fabricated’ generally refers to a structurethat has been specifically designed, engineered, and/or constructed soas to exist at a surface of the device and is not to be equated with asurface feature that is merely an incidental product of a deviceformation process. Thus, there will be a predetermined pattern ofnanostructures on the surface of the microneedles.

During use, the device, and specifically, the nano-sized structures onthe surface of the microneedles, may interact with the dermal tissue andcomponents thereof. This interaction may regulate or modulate (i.e.,change) intracellular and/or intercellular signal transductionassociated with cell/cell interactions, endocytosis, inflammatoryresponse, and so forth. For instance, through interaction between thenanotopography on a surface and surrounding biological materials orstructures, the device may regulate and/or modulate membrane potential,membrane proteins, and/or intercellular junctions (e.g., tightjunctions, gap junctions, and/or desmasomes). This may encourage thetransdermal delivery of the siRNA constructs. Moreover, the siRNAconstructs may be delivered across the dermal barrier withoutinstigating a foreign body or immune response.

The device may be constructed from a variety of materials, includingmetals, ceramics, semiconductors, organics, polymers, etc., as well ascomposites thereof. By way of example, pharmaceutical grade stainlesssteel, titanium, nickel, iron, gold, tin, chromium, copper, alloys ofthese or other metals, silicon, silicon dioxide, and polymers may beutilized in forming a device. Typically, the microneedles of the deviceare formed of a biocompatible material that is capable of carrying apattern of nano-sized structures on a surface. The term “biocompatible”generally refers to a material that does not substantially adverselyaffect the cells or tissues in the area where the device is to bedelivered. It is also intended that the materials do not cause anysubstantially medically undesirable effect in any other areas of aliving subject utilizing a device. Biocompatible materials may besynthetic or natural. Some examples of suitable biocompatible materials,which are also biodegradable, include polymers of hydroxy acids such aslactic acid and glycolic acid polylactide, polyglycolide,polylactide-co-glycolide, copolymers with PEG, polyanhydrides,poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valericacid), and poly(lactide-co-caprolactone). Other suitable materials mayinclude, without limitation, polycarbonate, polymethacrylic acid,ethylenevinyl acetate, polytetrafluorethylene, and polyesters. Thevarious components of a device (e.g., the microneedles, the base, thetop, drug contacting areas, etc.) may be non-porous or porous in nature,may be homogeneous or heterogeneous across the device with regard tomaterials, geometry, solidity, and so forth, and may have a rigid fixedor a semi-fixed shape.

FIG. 1 illustrates a typical microneedle transdermal device 10. As maybe seen, the device includes an array of individual needles 12; eachformed to a size and shape so as to penetrate all or a portion of thedermal barrier without breakage of the individual microneedles.Microneedles may be solid, as in FIG. 1, porous, or may include a hollowportion. A microneedle may include a hollow portion, e.g., an annularbore that may extend throughout all or a portion of the needle,extending parallel to the direction of the needle or branching orexiting at a side of the needle, as appropriate. For example, FIG. 2illustrates an array of microneedles 14 each including a channel 16 in aside of the needle as may be utilized for delivery of an siRNA constructto a subdermal location. For instance, a channel 16 may be in at leastpartial alignment with an aperture in base 15 so as to form a junctionbetween the aperture and channel 16 allowing the passage of a substancethrough the channel 16.

The dimensions of the channel 16, when present, may be specificallyselected to induce capillary flow of a drug compound. Capillary flowgenerally occurs when the adhesive forces of a fluid to the walls of achannel are greater than the cohesive forces between the liquidmolecules. Specifically, capillary pressure is inversely proportional tothe cross-sectional dimension of the channel 16 and directlyproportional to the surface tension of the liquid, multiplied by thecosine of the contact angle of the fluid in contact with the materialforming the channel. Thus, to facilitate capillary flow in the patch,the cross-sectional dimension (e.g., width, diameter, etc.) of thechannel 16 may be selectively controlled, with smaller dimensionsgenerally resulting in higher capillary pressure. For example, in someembodiments, the cross-sectional dimension of the channel typicallyranges from about 1 micrometer to about 100 micrometers, in someembodiments from about 5 micrometers to about 50 micrometers, and insome embodiments, from about 10 micrometers to about 30 micrometers. Thedimension may be constant or it may vary as a function of the length ofthe channel 16. The length of the channel may also vary to accommodatedifferent volumes, flow rates, and dwell times for the drug compound.For example, the length of the channel may be from about 10 micrometersto about 800 micrometers, in some embodiments from about 50 micrometersto about 500 micrometers, and in some embodiments, from about 100micrometers to about 300 micrometers. The cross-sectional area of thechannel may also vary. For example, the cross-sectional area may be fromabout 50 square micrometers to about 1,000 square micrometers, in someembodiments from about 100 square micrometers to about 500 squaremicrometers, and in some embodiments, from about 150 square micrometersto about 350 square micrometers. Further, the aspect ratio(length/cross-sectional dimension) of the channel may range from about 1to about 50, in some embodiments from about 5 to about 40, and in someembodiments from about 10 to about 20. In cases where thecross-sectional dimension (e.g., width, diameter, etc.) and/or lengthvary as a function of length, the aspect ratio can be determined fromthe average dimensions.

It should be understood that the number of microneedles shown in thefigures is for illustrative purposes only. The actual number ofmicroneedles used in a microneedle assembly may, for example, range fromabout 500 to about 10,000, in some embodiments from about 2,000 to about8,000, and in some embodiments, from about 4,000 to about 6,000.

An individual microneedle may have a straight or a tapered shaft. In oneembodiment, the diameter of a microneedle may be greatest at the baseend of the microneedle and taper to a point at the end distal the base.A microneedle may also be fabricated to have a shaft that includes botha straight (untapered) portion and a tapered portion.

A microneedle may be formed with a shaft that is circular ornon-circular in cross-section. For example, the cross-section of amicroneedle may be polygonal (e.g. star-shaped, square, triangular),oblong, or any other shape. The shaft may have one or more bores and/orchannels.

The size of individual needles may be optimized depending upon thedesired targeting depth, the strength requirements of the needle toavoid breakage in a particular delivery location, etc. For instance, thecross-sectional dimension of a transdermal microneedle may be betweenabout 10 nanometers (nm) and 1 millimeter (mm), or between about 1micrometer (μm) and about 200 micrometers, or between about 10micrometers and about 100 micrometers. The outer diameter may be betweenabout 10 micrometers and about 100 micrometers and the inner diameter ofa hollow needle may be between about 3 micrometers and about 80micrometers. The tip typically has a radius that is less than or equalto about 1 micrometer.

The length of a microneedle will generally depend upon the desiredapplication. For instance, a microneedle may be between about 1micrometer and about 1 millimeter in length, for instance about 500micrometers or less, or between about 10 micrometers and about 500micrometers, or between about 30 micrometers and abut 200 micrometers.

An array of microneedles need not include microneedles that are allidentical to one another. An array may include a mixture of microneedleshaving various lengths, outer diameters, inner diameters,cross-sectional shapes, nanostructured surfaces, and/or spacings betweenthe microneedles. For example, the microneedles may be spaced apart in auniform manner, such as in a rectangular or square grid or in concentriccircles. The spacing may depend on numerous factors, including heightand width of the microneedles, as well as the amount and type of anysubstance that is intended to be moved through the microneedles. While avariety of arrangements of microneedles is useful, a particularly usefularrangement of microneedles is a “tip-to-tip” spacing betweenmicroneedles of about 50 micrometers or more, in some embodiments about100 to about 800 micrometers, and in some embodiments, from about 200 toabout 600 micrometers.

Referring again to FIG. 1, microneedles may be held on a substrate 20(i.e., attached to or unitary with a substrate) such that they areoriented perpendicular or at an angle to the substrate. In oneembodiment, the microneedles may be oriented perpendicular to thesubstrate and a larger density of microneedles per unit area ofsubstrate may be provided. However, an array of microneedles may includea mixture of microneedle orientations, heights, materials, or otherparameters. The substrate 20 may be constructed from a rigid or flexiblesheet of metal, ceramic, plastic or other material. The substrate 20 mayvary in thickness to meet the needs of the device, such as about 1000micrometers or less, in some embodiments from about 1 to about 500micrometers, and in some embodiments, from about 10 to about 200micrometers.

The device may define a nanotopography on the surface of a microneedlein a random or organized pattern. The device may additionally define ananotopography on the substrate surface from which the microneedleextends, though this is not a requirement. FIG. 3 schematicallyillustrates the ends of two representative microneedles 22. Microneedles22 define a central bore 24 as may be used for delivery of an siRNAconstruct via the microneedles 22. The surface 25 of microneedle 22 maydefine nanotopography 26. In this particular embodiment, thenanotopography 26 defines a random pattern on the surface 25 of themicroneedle 22.

A microneedle may include a plurality of identical structures formed ona surface or may include different structures formed of various sizes,shapes and combinations thereof. A predetermined pattern of structuresmay include a mixture of structures having various lengths, diameters,cross-sectional shapes, and/or spacings between the structures. Forexample, the structures may be spaced apart in a uniform manner, such asin a rectangular or square grid or in concentric circles. In oneembodiment, structures may vary with regard to size and/or shape and mayform a complex nanotopography. In one embodiment a complexnanotopography may define a fractal or fractal-like geometry.

As utilized herein, the term “fractal” generally refers to a geometricor physical structure having a fragmented shape at all scales ofmeasurement between a greatest and a smallest scale such that certainmathematical or physical properties of the structure behave as if thedimensions of the structure are greater than the spatial dimensions.Mathematical or physical properties of interest may include, forexample, the perimeter of a curve or the flow rate in a porous medium.The geometric shape of a fractal may be split into parts, each of whichdefines self-similarity. Additionally, a fractal has a recursivedefinition and has a fine structure at arbitrarily small scales.

As utilized herein, the term “fractal-like” generally refers to ageometric or physical structure having one or more, but not all, of thecharacteristics of a fractal. For instance, a fractal-like structure mayinclude a geometric shape that includes self-similar parts, but may notinclude a fine structure at an arbitrarily small scale. In anotherexample, a fractal-like geometric shape or physical structure may notdecrease (or increase) in scale equally between iterations of scale, asmay a fractal, though it will increase or decrease between recursiveiterations of a geometric shape of the pattern. A fractal-like patternmay be simpler than a fractal. For example, it may be regular andrelatively easily described in traditional Euclidean geometric language,whereas a fractal may not.

A microneedle surface defining a complex nanotopography may includestructures of the same general shape (e.g., pillars) and the pillars maybe formed to different scales of measurement (e.g., nano-scale pillarsas well as micro-scale pillars). In another embodiment, a microneedlemay include at a surface structures that vary in both scale size andshape or that vary only in shape while formed to the same nano-sizedscale. Additionally, structures may be formed in an organized array orin a random distribution. In general, at least a portion of thestructures may be nanostructures formed on a nano-sized scale, e.g.,defining a cross-sectional dimension of less than about 500 nm, forinstance less than about 400 nm, less than about 250 nm, or less thanabout 100 nm. The cross sectional dimension of the nanostructures maygenerally be greater than about 5 nanometers, for instance greater thanabout 10 nanometers, or greater than about 20 nanometers. For example,the nanostructures may define a cross sectional dimension between about5 nanometers and about 500 nanometers, between about 20 nanometers andabout 400 nanometers, or between about 100 nanometers and about 300nanometers. In cases where the cross sectional dimension of ananostructure varies as a function of height of the nanostructure, thecross sectional dimension can be determined as the average from the baseto the tip of the nanostructures, or as the maximum cross sectionaldimension of the structure, for example the cross sectional dimension atthe base of a cone-shaped nanostructure.

FIG. 4 illustrates one embodiment of a complex nanotopography as may beformed on a surface. This particular pattern includes a central largepillar 100 and surrounding pillars 102, 104, of smaller dimensionsprovided in a regular pattern. As may be seen, this pattern includes aniteration of pillars, each of which is formed with the same generalshape, but vary with regard to horizontal dimension. This particularcomplex pattern is an example of a fractal-like pattern that does notinclude identical alteration in scale between successive recursiveiterations. For example, while the pillars 102 are first nanostructuresthat define a horizontal dimension that is about one third that of thelarger pillar 100, which is a microstructure, the pillars 104 are secondnanostructures that define a horizontal dimension that is about one halfthat of the pillars 102.

A pattern that includes structures of different sizes may include largerstructures having a cross-sectional dimension formed on a larger scale,e.g., microstructures having a cross-sectional dimension greater thanabout 500 nanometers in combination with smaller nanostructures. In oneembodiment, microstructures of a complex nanotopography may have across-sectional dimension between about 500 nanometers and about 10micrometers, between about 600 nanometers and about 1.5 micrometers, orbetween about 650 nanometers and about 1.2 micrometers. For example, thecomplex nanotopography of FIG. 4 includes micro-sized pillars 100 havinga cross sectional dimension of about 1.2 micrometers.

When a pattern includes one or more larger microstructures, forinstance, having a cross-sectional dimension greater than about 500nanometers, determined either as the average cross sectional dimensionof the structure or as the largest cross sectional dimension of thestructure, the complex nanotopography will also include nanostructures,e.g., first nanostructures, second nanostructures of a different sizeand/or shape, etc. For example, pillars 102 of the complexnanotopography of FIG. 4 have a cross-sectional dimension of about 400nanometers, and pillars 104 have a cross-sectional dimension of about200 nanometers.

A nanotopography may be formed of any number of different elements. Forinstance, a pattern of elements may include two different elements,three different elements, an example of which is illustrated in FIG. 4,four different elements, or more. The relative proportions of therecurrence of each different element may also vary. In one embodiment,the smallest elements of a pattern will be present in larger numbersthan the larger elements. For instance in the pattern of FIG. 4, thereare eight pillars 104 for each pillar 102, and there are eight pillars102 for the central large pillar 100. As elements increase in size,there may generally be fewer recurrences of the element in thenanotopography. By way of example, a first element that is about 0.5,for instance between about 0.3 and about 0.7 in cross-sectionaldimension as a second, larger element may be present in the topographyabout five times or more than the second element. A first element thatis approximately 0.25, or between about 0.15 and about 0.3 incross-sectional dimension as a second, larger element may be present inthe topography about 10 times or more than the second element.

The spacing of individual elements may also vary. For instance,center-to-center spacing of individual structures may be between about50 nanometers and about 1 micrometer, for instance between about 100nanometers and about 500 nanometers. For example, center-to-centerspacing between structures may be on a nano-sized scale. For instance,when considering the spacing of nano-sized structures, thecenter-to-center spacing of the structures may be less than about 500nanometers. This is not a requirement of a topography, however, andindividual structures may be farther apart. The center-to-center spacingof structures may vary depending upon the size of the structures. Forexample, the ratio of the average of the cross-sectional dimensions oftwo adjacent structures to the center-to-center spacing between thosetwo structures may be between about 1:1 (e.g., touching) and about 1:4,between about 1:1.5 and about 1:3.5, or between about 1:2 and about 1:3.For instance, the center to center spacing may be approximately doublethe average of the cross-sectional dimensions of two adjacentstructures. In one embodiment, two adjacent structures each having across-sectional dimension of about 200 nanometers may have acenter-to-center spacing of about 400 nanometers. Thus, the ratio of theaverage of the diameters to the center-to-center spacing in this case is1:2.

Structure spacing may be the same, i.e., equidistant, or may vary forstructures in a pattern. For instance, the smallest structures of apattern may be spaced apart by a first distance, and the spacing betweenthese smallest structures and a larger structure of the pattern orbetween two larger structures of the pattern may be the same ordifferent as this first distance.

For example, in the pattern of FIG. 4, the smallest structures 104 havea center-to-center spacing of about 200 nanometers. The distance betweenthe larger pillars 102 and each surrounding pillar 104 is less, about100 nanometers. The distance between the largest pillar 100 and eachsurrounding pillar 104 is also less than the center-to-center spacingbetween to smallest pillars 104, about 100 nanometers. Of course, thisis not a requirement, and all structures may be equidistant from oneanother or any variation in distances. In one embodiment, differentstructures may be in contact with one another, for instance atop oneanother, as discussed further below, or adjacent one another and incontact with one another.

Structures of a topography may all be formed to the same height,generally between about 10 nanometers and about 1 micrometer, but thisis not a requirement, and individual structures of a pattern may vary insize in one, two, or three dimensions. In one embodiment, some or all ofthe structures of a topography can have a height of less than about 20micrometers, less than about 10 micrometers, or less than about 1micrometer, for instance less than about 750 nanometers, less than about680 nanometers, or less than about 500 nanometers. For instance thestructures can have a height between about 50 nanometers and about 20micrometers or between about 100 nanometers and about 700 nanometers.For example, nanostructures or microstructures can have a height betweenabout 20 nm and about 500 nm, between about 30 nm and about 300 nm, orbetween about 100 nm and about 200 nm, though it should be understoodthat structures may be nano-sized in a cross sectional dimension and mayhave a height that may be measured on a micro-sized scale, for instancegreater than about 500 nm. Micro-sized structures can have a height thatis the same or different from nano-sized structures of the same pattern.For instance, micro-sized structures can have a height of between about500 nanometers and about 20 micrometers, or between about 1 micrometerand about 10 micrometers, in another embodiment. Micro-sized structuresmay also have a cross sectional dimension on a micro-scale greater thanabout 500 nm, and may have a height that is on a nano-sized scale ofless than about 500 nm.

The aspect ratio of the structures (the ratio of the height of astructure to the cross sectional dimension of the structure) can bebetween about 0.15 and about 30, between about 0.2 and about 5, betweenabout 0.5 and about 3.5, or between about 1 and about 2.5. For instance,nanostructures may have an aspect ratio falling within any of theseranges.

The device surface may include a single instance of a pattern, as shownin FIG. 4, or may include multiple iterations of the same or differentpatterns. For example, FIG. 5 illustrates a surface pattern includingthe pattern of FIG. 4 in multiple iterations over a surface.

The formation of nanotopography on a microneedle surface may increasethe surface area of the microneedle without a corresponding increase involume. Increase in the surface area to volume ratio is believed toimprove the interaction of the microneedle surface with surroundingbiological materials. For instance, increase in the surface area tovolume ratio is believed to encourage mechanical interaction between thenanotopography and surrounding proteins, e.g., extracellular matrix(ECM) proteins and/or plasma membrane proteins. As utilized herein, theterm “protein” generally refers to a molecular chain of amino acids thatis capable of interacting structurally, enzymatically or otherwise withother proteins, polypeptides or any other organic or inorganic molecule.

In general, the surface area to volume ratio of a nanopatterned surfacemay be greater than about 10,000 cm⁻¹, greater than about 150,000 cm⁻¹,or greater than about 750,000 cm⁻¹. Determination of the surface area tovolume ratio may be carried out according to any standard methodology asis known in the art. For instance, the specific surface area of asurface may be obtained by the physical gas adsorption method (B.E.T.method) with nitrogen as the adsorption gas, as is generally known inthe art and described by Brunauer, Emmet, and Teller (J. Amer. Chem.Soc., vol. 60, February, 1938, pp. 309-319), incorporated herein byreference. The BET surface area can be less than about 5 m²/g, in oneembodiment, for instance between about 0.1 m²/g and about 4.5 m²/g, orbetween about 0.5 m²/g and about 3.5 m²/g. Values for surface area andvolume may also be estimated from the geometry of molds used to form asurface, according to standard geometric calculations. For example, thevolume may be estimated according to the calculated volume for eachpattern element and the total number of pattern elements in a givenarea, e.g., over the surface of a single microneedle.

For a device that defines a fractal or fractal-like patternednanotopography at a surface, the nanotopography may be characterizedthrough determination of the fractal dimension of the pattern. Thefractal dimension is a statistical quantity that gives an indication ofhow completely a fractal appears to fill space as the recursiveiterations continue to smaller and smaller scale. The fractal dimensionof a two dimensional structure may be represented as:

$D = \frac{\log\;{N(e)}}{\log(e)}$

where N(e) is the number of self-similar structures needed to cover thewhole object when the object is reduced by 1/e in each spatialdirection.

For example, when considering the 2 dimensional fractal known as theSierpenski triangle illustrated in FIG. 6, in which the mid-points ofthe three sides of an equilateral triangle are connected and theresulting inner triangle is removed, the fractal dimension is calculatedas follows:

$D = \frac{\log\;{N(e)}}{\log(e)}$ $D = \frac{\log\; 3}{\log\; 2}$D ≈ 1.585

Thus, the Sierpenski triangle fractal exhibits an increase in linelength over the initial two dimensional equilateral triangle.Additionally, this increase in line length is not accompanied by acorresponding increase in area.

The fractal dimension of the pattern illustrated in FIG. 4 isapproximately 1.84. In one embodiment, nanotopography of a surface ofthe device may exhibit a fractal dimension of greater than about 1, forinstance between about 1.2 and about 5, between about 1.5 and about 3,or between about 1.5 and about 2.5.

FIGS. 7A and 7B illustrate increasing magnification images of anotherexample of a complex nanotopography. The nanotopography of FIGS. 7A and7B includes an array of fibrous-like pillars 70 located on a substrate.At the distal end of each individual pillar, the pillar splits intomultiple smaller fibers 60. At the distal end of each of these smallerfibers 60, each fiber splits again into multiple filaments (not visiblein FIGS. 7A and 7B). Structures formed on a surface that have an aspectratio greater than about 1 may be flexible, as are the structuresillustrated in FIGS. 7A and 7B, or may be stiff.

FIGS. 7C and 7D illustrate another example of a complex nanotopography.In this embodiment, a plurality of pillars 72 each including an annularhollow therethrough 71 are formed on a substrate. At the distal end ofeach hollow pillar, a plurality of smaller pillars 62 is formed. As maybe seen, the pillars of FIGS. 7C and 7D maintain their stiffness andupright orientation. Additionally, and in contrast to previous patterns,the smaller pillars 62 of this embodiment differ in shape from thelarger pillars 72. Specifically, the smaller pillars 62 are not hollow,but are solid. Thus, nanotopography including structures formed to adifferent scale need not have all structures formed with the same shape,and structures may vary in both size and shape from the structures of adifferent scale.

FIG. 8 illustrates another pattern including nano-sized structures asmay be formed on a microneedle surface. As may be seen, in thisembodiment, individual pattern structures may be formed at the samegeneral size, but with different orientations and shapes from oneanother.

In addition to or alternative to the examination of surface area tovolume ratio and/or fractal dimension, the microneedles of the siRNAdelivery devices may be characterized by other methods including,without limitation, surface roughness, elastic modulus, and surfaceenergy.

Methods for determining the surface roughness are generally known in theart. For instance, an atomic force microscope process in contact ornon-contact mode may be utilized according to standard practice todetermine the surface roughness of a material. Surface roughness thatmay be utilized to characterize a microneedle may include the averageroughness (R_(A)), the root mean square roughness, the skewness, and/orthe kurtosis. In general, the average surface roughness (i.e., thearithmetical mean height of the surface are roughness parameter asdefined in the ISO 25178 series) of a surface defining a fabricatednanotopography thereon may be less than about 200 nanometers, less thanabout 190 nanometers, less than about 100 nanometers, or less than about50 nanometers. For instance, the average surface roughness may bebetween about 10 nanometers and about 200 nanometers, or between about50 nanometers and about 190 nanometers.

The microneedle surface may be characterized by the elastic modulus ofthe surface, for instance by the change in elastic modulus upon theaddition of a nanotopography to the surface. In general, the addition ofa plurality of structures forming nanotopography on the microneedlesurface may decrease the elastic modulus of a material, as the additionof nano-sized structures on the surface will lead to a reduction incontinuity of the surface and a related change in surface area. Ascompared to a similar microneedle formed according to the same processand of the same materials, but for the pattern of nanotopography on thesurface, a microneedle including nanotopography thereon may exhibit adecrease in elastic modulus of between about 35% and about 99%, forinstance between about 50% and about 99%, or between about 75% and about80%. By way of example, the effective compression modulus of ananopatterned surface can be less than about 50 MPa, or less than about20 MPa. In one embodiment the effective compression modulus can bebetween about 0.2 MPa and about 50 MPa, between about 5 MPa and about 35MPa, or between about 10 MPa and about 20 MPa. The effective shearmodulus can be less than about 320 MPa, or less than about 220 MPa. Forinstance, the effective shear modulus can be between about 4 MPa andabout 320 MPa, or between about 50 MPa and about 250 MPa, in oneembodiment.

A microneedle including nanotopography thereon may also exhibit anincrease in surface energy as compared to a similar microneedle thatdoes not have the pattern of nanotopography thereon. For instance, themicroneedle including a nanotopography formed thereon may exhibit anincrease in surface energy as compared to a similar microneedle of thesame materials and formed according to the same methods, but for theinclusion of the pattern of nanotopography on the surface. For instance,the water contact angle of a surface including a nanotopography thereoncan be greater than about 80°, greater than about 90°, greater thanabout 100°, or greater than about 110°. For example, the water contactangle of a surface can be between about 80° and about 150°, betweenabout 90° and about 130°, or between about 100° and about 120°, in oneembodiment.

When forming nanostructures on the surface of the device, the packingdensity of the structures may be maximized. For instance, square packing(FIG. 9A), hexagonal packing (FIG. 9B), or some variation thereof may beutilized to pattern the elements on the microneedle. When designing apattern in which various sized elements of cross sectional areas A, B,and C are adjacent to one another on the microneedle, circle packing asindicated in FIG. 9C may be utilized. Of course, variations in packingdensity and determination of associated alterations in characteristicsof the surface are well within the abilities of one of skill in the art.

The microneedles including a fabricated nanotopography on the surface ofthe microneedles may be formed according to a single-step process, i.e.,the microneedles are formed with the nanostructures on the surface atthe time of formation. Alternatively, a multi-step process may be used,in which a pattern of nanostructures are fabricated on a pre-formedmicroneedle. For example, an array of microneedles may be first formedand then a random or non-random pattern of nanostructures may befabricated on the surface of the formed microneedles. In either thesingle-step or two-step process, the nano-sized structures may befabricated on the microneedle surface or on a mold surface according toany suitable nanotopography fabrication method including, withoutlimitation, nanoimprinting, injection molding, lithography, embossingmolding, and so forth.

An array of microneedles may be formed according to any standardmicrofabrication technique including, without limitation, lithography;etching techniques, such as wet chemical, dry, and photoresist removal;thermal oxidation of silicon; electroplating and electroless plating;diffusion processes, such as boron, phosphorus, arsenic, and antimonydiffusion; ion implantation; film deposition, such as evaporation(filament, electron beam, flash, and shadowing and step coverage),sputtering, chemical vapor deposition (CVD), epitaxy (vapor phase,liquid phase, and molecular beam), electroplating, screen printing, andlamination; stereolithography; laser machining; and laser ablation(including projection ablation).

An electrochemical etching process may be utilized in whichelectrochemical etching of solid silicon to porous silicon is used tocreate extremely fine (on the order of 0.01 μm) silicon networks thatmay be used as piercing structures. This method may use electrolyticanodization of silicon in aqueous hydrofluoric acid, potentially incombination with light, to etch channels into the silicon. By varyingthe doping concentration of the silicon wafer to be etched, theelectrolytic potential during etching, the incident light intensity, andthe electrolyte concentration, control over the ultimate pore structuremay be achieved. The material not etched (i.e. the silicon remaining)forms the microneedles.

Plasma etching may also be utilized, in which deep plasma etching ofsilicon is carried out to create microneedles with diameters on theorder of 0.1 μm or larger. Needles may be fabricated indirectly bycontrolling the voltage (as in electrochemical etching).

Lithography techniques, including photolithography, e-beam lithography,X-ray lithography, and so forth may be utilized for primary patterndefinition and formation of a master die. Replication may then becarried out to form a device including an array of microneedles. Commonreplication methods include, without limitation, solvent-assistedmicromolding and casting, embossing molding, injection molding, and soforth. Self-assembly technologies including phase-separated blockcopolymer, polymer demixing and colloidal lithography techniques mayalso be utilized in forming a nanotopography on a surface.

Combinations of methods may be used, as is known. For instance,substrates patterned with colloids may be exposed to reactive ionetching (RIE, also known as dry etching) so as to refine thecharacteristics of a fabricated nanostructure such as nanopillardiameter, profile, height, pitch, and so forth. Wet etching may also beemployed to produce alternative profiles for fabricated nanostructuresinitially formed according to a different process, e.g., polymerde-mixing techniques.

Structure diameter, shape, and pitch may be controlled via selection ofappropriate materials and methods. For example, etching of metalsinitially evaporated onto colloidal-patterned substrates followed bycolloidal lift-off generally results in prism-shaped pillars. An etchingprocess may then be utilized to complete the structures as desired.Ordered non-spherical polymeric nanostructures may also be fabricatedvia temperature-controlled sintering techniques, which form a variety ofordered trigonal nanometric features in colloidal interstices followingselective dissolution of polymeric nanoparticles. These and othersuitable formation processes are generally known in the art (see, e.g.,Wood, J R Soc Interface, 2007 February 22; 4(12): 1-17, incorporatedherein by reference).

Other methods as may be utilized in forming a microneedle including afabricated nanotopography on a surface include nanoimprint lithographymethods utilizing ultra-high precision laser machining techniques,examples of which have been described by Hunt, et al. (U.S. Pat. No.6,995,336) and Guo, et al. (U.S. Pat. No. 7,374,864), both of which areincorporated herein by reference. Nanoimprint lithography is anano-scale lithography technique in which a hybrid mold is utilizedwhich acts as both a nanoimprint lithography mold and a photolithographymask. A schematic of a nanoimprint lithography technique is illustratedin FIGS. 10A-10C. During fabrication, a hybrid mold 30 imprints into asubstrate 32 via applied pressure to form features (e.g., microneedlesdefining nanotopography) on a resist layer (FIG. 10A). In general, thesurface of the substrate 32 may be heated prior to engagement with themold 30 to a temperature above its glass transition temperature (T_(g)).While the hybrid mold 30 is engaged with the substrate 32, a flow ofviscous polymer may be forced into the mold cavities to form features 34(FIG. 10B). The mold and substrate may then be exposed to ultravioletlight. The hybrid mold is generally transmissive to UV radiation savefor certain obstructed areas. Thus, the UV radiation passes throughtransmissive portions and into the resist layer. Pressure is maintainedduring cooling of the mold and substrate. The hybrid mold 30 is thenremoved from the cooled substrate 32 at a temperature below T_(g) of thesubstrate and polymer (FIG. 10C).

To facilitate the release of the nanoimprinted substrate 32 includingfabricated features 34 from the mold 30, as depicted in FIG. 10C, it isadvantageous to treat the mold 30 with a low energy coating to reducethe adhesion with the substrate 32, as a lower surface energy of themold 30 and the resulting greater surface energy difference between themold 30, substrate 32, and polymer may ease the release between thematerials. By way of example, a silicon mold coating may be used such astrideca-(1,1,2,2-tetrahydro)-octytrichloro silane (F₁₃-TCS).

A nanoimprinting process is a dynamic one which includes filling a moldfollowed by detachment of a formed polymer from the mold. To fill themold features, the polymer temperature must be raised to a level highenough to initiate flow under the applied pressure. The higher thetemperature, the lower the polymer viscosity, and the faster and easierthe mold will fill. A higher pressure will also improve the fill rateand overall fill for better mold replication. To release thenanoimprinted substrate from the mold, the substrate temperature may belowered to a point where the yield strength exceeds the adhesionalforces exerted by the mold. By varying the temperature it is alsopossible to draw the polymer features during detachment to obtaindifferent structures, for instance structures as illustrated in FIG. 8.

The nanostructures may also be formed on the microneedle according tochemical addition processes. For instance, film deposition, sputtering,chemical vapor deposition (CVD), epitaxy (vapor phase, liquid phase, andmolecular beam), electroplating, and so forth may be utilized forbuilding structures on a surface.

Self-assembled monolayer processes as are known in the art may beutilized to form the structures on the microneedle surface. Forinstance, the ability of block copolymers to self-organize may be usedto form a monolayer pattern on the surface. The pattern may then be usedas a template for the growth of the desired structures, e.g., colloids,according to the pattern of the monolayer.

By way of example, a two-dimensional, cross-linked polymer network maybe produced from monomers with two or more reactive sites. Suchcross-linked monolayers have been made using self-assembling monolayer(SAM) (e.g., a gold/alkyl thiol system) or Langmuir-Blodgett (LB)monolayer techniques (Ahmed et al., Thin Solid Films 187: 141-153(1990)) as are known in the art. The monolayer may be crosslinked, whichmay lead to formation of a more structurally robust monolayer.

The monomers used to form the patterned monolayer may incorporate allthe structural moieties necessary to affect the desired polymerizationtechnique and/or monolayer formation technique, as well as to influencesuch properties as overall solubility, dissociation methods, andlithographic methods. A monomer may contain at least one, and more oftenat least two, reactive functional groups.

A molecule used to form an organic monolayer may include any of variousorganic functional groups interspersed with chains of methylene groups.For instance a molecule may be a long chain carbon structure containingmethylene chains to facilitate packing. The packing between methylenegroups may allow weak Van der Waals bonding to occur, enhancing thestability of the monolayer produced and counteracting the entropicpenalties associated with forming an ordered phase. In addition,different terminal moieties, such as hydrogen-bonding moieties, may bepresent at one terminus of the molecules, in order to allow growth ofstructures on the formed monolayer, in which case the polymerizablechemical moieties may be placed in the middle of the chain or at theopposite terminus. Any suitable molecular recognition chemistry may beused in forming the assembly. For instance, structures may be assembledon a monolayer based on electrostatic interaction, Van der Waalsinteraction, metal chelation, coordination bonding (i.e., Lewisacid/base interactions), ionic bonding, covalent bonding, or hydrogenbonding.

When utilizing a SAM-based system, an additional molecule may beutilized to form the template. This additional molecule may haveappropriate functionality at one of its termini in order to form a SAM.For example, on a gold surface, a terminal thiol may be included. Thereare a wide variety of organic molecules that may be employed to effectreplication. Topochemically polymerizable moieties, such as dienes anddiacetylenes, are particularly desirable as the polymerizing components.These may be interspersed with variable lengths of methylene linkers.

For an LB monolayer, only one monomer molecule is needed because themolecular recognition moiety may also serve as the polar functionalgroup for LB formation purposes. Lithography may be carried out on a LBmonolayer transferred to a substrate, or directly in the trough. Forexample, an LB monolayer of diacetylene monomers may be patterned by UVexposure through a mask or by electron beam patterning.

Monolayer formation may be facilitated by utilizing molecules thatundergo a topochemical polymerization in the monolayer phase. Byexposing the assembling film to a polymerization catalyst, the film maybe grown in situ, and changed from a dynamic molecular assembly to amore robust polymerized assembly.

Any of the techniques known in the art for monolayer patterning may beused. Techniques useful in patterning the monolayer include, but are notlimited to, photolithography, e-beam techniques, focused ion-beamtechniques, and soft lithography. Various protection schemes such asphotoresist may be used for a SAM-based system. Likewise, blockcopolymer patterns may be formed on gold and selectively etched to formpatterns. For a two-component system, patterning may also be achievedwith readily available techniques.

Soft lithography techniques may be utilized to pattern the monolayer inwhich ultraviolet light and a mask may be used for patterning. Forinstance, an unpatterned base monolayer may be used as a platform forassembly of a UV/particle beam reactive monomer monolayer. The monomermonolayer may then be patterned by UV photolithography, e-beamlithography, or ion beam lithography, even though the base SAM is notpatterned.

Growth of structures on a patterned monolayer may be achieved by variousgrowth mechanisms, such as through appropriate reduction chemistry of ametal salt and the use of seed or template-mediated nucleation. Usingthe recognition elements on the monolayer, inorganic growth may becatalyzed at this interface by a variety of methods. For instanceinorganic compounds in the form of colloids bearing the shape of thepatterned organic monolayer may be formed. For instance calciumcarbonate or silica structures may be templated by various carbonylfunctionalities such as carboxylic acids and amides. By controlling thecrystal growth conditions, it is possible to control the thickness andcrystal morphology of the mineral growth. Titanium dioxide may also betemplated.

Templated electroless plating techniques may be used to synthesizemetals using existing organic functional groups. In particular, bychelating metal atoms to the carbonyl moieties of the organic pattern,electroless metal deposition may be catalyzed on the pattern, formingpatterned metallic colloids. For instance, Cu, Au, Ni, Ag, Pd, Pt andmany other metals plateable by electroless plating conditions may beused to form metal structures in the shape of the organic monolayer. Bycontrolling the electroless plating conditions, it is possible tocontrol the thickness of the plated metal structures.

Other ‘bottom-up’ type growth methods as are known in the art may beutilized, for example a method as described in U.S. Pat. No. 7,189,435to Tuominen, et al., which is incorporated herein by reference, may beutilized. According to this method, a conducting or semiconductingsubstrate (for example, a metal, such as gold) may be coated with ablock copolymer film (for example, a block copolymer ofmethylmethacrylate and styrene), where one component of the copolymerforms nanoscopic cylinders in a matrix of another component of thecopolymer. A conducting layer may then be placed on top of the copolymerto form a composite structure. Upon vertical orientation of thecomposite structure, some of the first component may be removed, forinstance by exposure to UV radiation, an electron beam, or ozone,degradation, or the like to form nanoscopic pores in that region of thesecond component.

In another embodiment, described in U.S. Pat. No. 6,926,953 to Nealey,et al., incorporated herein by reference, copolymer structures may beformed by exposing a substrate with an imaging layer thereon, forinstance an alkylsiloxane or an octadecyltrichlorosilane self assembledmonolayer, to two or more beams of selected wavelengths to forminterference patterns at the imaging layer to change the wettability ofthe imaging layer in accordance with the interference patterns. A layerof a selected block copolymer, for instance a copolymer of polystyreneand poly(methyl methacrylate) may then be deposited onto the exposedimaging layer and annealed to separate the components of the copolymerin accordance with the pattern of wettability and to replicate thepattern of the imaging layer in the copolymer layer. Stripes or isolatedregions of the separated components may thus be formed with periodicdimensions in the range of 100 nm or less.

The microneedle surface may include a random distribution of fabricatednanostructures. Optionally, the microneedle surface may includeadditional materials, in conjunction with the fabricated nanostructures.For example, the microneedle may have fabricated thereon an electrospunfibrous layer, and a random or non-random pattern of nanostructures maybe fabricated on this electrospun layer.

Electrospinning includes of the use of a high voltage supplier to applyan electrical field to a polymer melt or solution held in a capillarytube, inducing a charge on the individual polymer molecules. Uponapplication of the electric field, a charge and/or dipolar orientationwill be induced at the air-surface interface. The induction causes aforce that opposes the surface tension. At critical field strength, theelectrostatic forces will overcome surface tension forces, and a jet ofpolymer material will be ejected from the capillary tube toward aconductive, grounded surface. The jet is elongated and accelerated bythe external electric field as it leaves the capillary tube. As the jettravels in air, some of the solvent may evaporate, leaving behindcharged polymer fibers which may be collected on the surface. As thefibers are collected, the individual and still wet fibers may adhere toone another, forming a nonwoven web on the surface. A pattern ofnanostructures may then be fabricated on the electrospun surface, forinstance through an embossing technique utilizing a mold defining thedesired nanostructures. Applying the mold to the microneedle surface atsuitable temperature and pressure may transfer the pattern to themicroneedle surface. A surface of random electrospun nano-sized fibersmay further improve the desirable characteristics of a microneedlesurface, e.g., one or more of surface area to volume ratio, surfaceroughness, surface energy, and so forth, and may provide associatedbenefits.

In addition to the nanostructures, the microneedle surface may bechemically functionalized for improved interaction with tissues orindividual cells. For instance, one or more biomolecules such aspolynucleotides, polypeptides, entire proteins, polysaccharides, and thelike may be bound to the microneedle surface prior to use.

In some embodiments, the microneedle surface may include suitablereactivity such that additional desired functionality may spontaneouslyattach to the surface with no pretreatment of the surface necessary.However, in other embodiments, pretreatment of the structured surfaceprior to attachment of the desired compound may be carried out. Forinstance, reactivity of a structure surface may be increased throughaddition or creation of amine, carboxylic acid, hydroxy, aldehyde,thiol, or ester groups on the surface. In one representative embodiment,a microneedle surface including a pattern of nanotstructures formedthereon may be aminated through contact with an amine-containingcompound such as 3-aminopropyltriethoxy silane in order to increase theamine functionality of the surface and bind one or more biomolecules tothe surface via the added amine functionality.

Materials as may be desirably bound to the surface of a patterned devicemay include ECM proteins such as laminins, tropoelastin or elastin,Tropocollagen or collagen, fibronectin, and the like. Short polypeptidefragments may be bound to the surface of a patterned device such as anRGD sequence, which is part of the recognition sequence of integrinbinding to many ECM proteins. Thus, functionalization of a microneedlesurface with RGD may encourage interaction of the device with ECMproteins and further limit foreign body response to the device duringuse.

The siRNA construct for delivery via the device may be associatedtherewith according to any suitable methodology. For instance, atransdermal microneedle patch may be utilized for delivery of materialsbeneath the stratum corneum to the stratum spinosum or the stratumgerminativum, or even deeper into the dermis. The siRNA, either free inthe composition or held in a protected state within the composition, maybe contained on the patch or fed to the patch so as to be transportedacross the stratum corneum in association with the microneedle, e.g.,within the microneedle or at the surface of the microneedle.

The microneedle transdermal patch may include a reservoir, e.g., avessel, a porous matrix, etc., that may store the siRNA construct andprovide the siRNA construct for delivery. The device may include areservoir within the device itself. For instance, the device may includea hollow, or multiple pores that may carry one or more siRNA constructsfor delivery. The siRNA construct may be released from the device viadegradation of a portion or the entire device or via diffusion of theagent from the device.

FIGS. 11A and 11B are perspective views of a device including areservoir. The device 110 includes a reservoir 112 defined by animpermeable backing layer 114 and a microneedle array 116. The backinglayer and the microneedle array 116 are joined together about the outerperiphery of the device, as indicated at 118. The impermeable backinglayer 114 may be joined by an adhesive, a heat seal or the like. Thedevice 110 also includes a plurality of microneedles 120. A releaseliner 122 may be removed prior to use of the device to exposemicroneedles 120.

A formulation including one or more siRNA constructs may be retainedwithin the reservoir 112. Materials suitable for use as impermeablebacking layer 114 may include materials such as polyesters,polyethylene, polypropylene and other synthetic polymers. The materialis generally heat or otherwise sealable to the backing layer to providea barrier to transverse flow of reservoir contents.

Reservoir 112, defined by the space or gap between the impermeablebacking layer 14 and the microneedle array 16, provides a storagestructure in which to retain the suspension of siRNA constructs to beadministered. The reservoir may be formed from a variety of materialsthat are compatible with an agent to be contained therein. By way ofexample, natural and synthetic polymers, metals, ceramics, semiconductormaterials, and composites thereof may form the reservoir.

In one embodiment, the reservoir may be attached to the substrate uponwhich the microneedles are located. According to another embodiment, thereservoir may be separate and removably connectable to the microneedlearray or in fluid communication with the microneedle array, for instancevia appropriate tubing, leur locks, etc.

The device may include one or a plurality of reservoirs for storingagents to be delivered. For instance, the device may include a singlereservoir that stores a single or multiple siRNA construct-containingformulation, or the device may include multiple reservoirs, each ofwhich stores one or more agents for delivery to all or a portion of thearray of microneedles. Multiple reservoirs may each store a differentmaterial that may be combined for delivery. For instance, a firstreservoir may contain an siRNA construct and a second reservoir maycontain a vehicle, e.g., saline, or a second siRNA construct. Thedifferent agents may be mixed prior to delivery. Mixing may be triggeredby any means, including, for example, mechanical disruption (i.e.puncturing, degradation, or breaking), changing the porosity, orelectrochemical degradation of the walls or membranes separating thechambers. Multiple reservoirs may contain different active agents fordelivery that may be delivered in conjunction with one another orsequentially.

In one embodiment, the reservoir may be in fluid communication with oneor more microneedles of the transdermal device, and the microneedles maydefine a structure (e.g., a central or lateral bore) to allow transportof delivered agents beneath the barrier layer.

In alternative embodiments, a device may include a microneedle assemblyand a reservoir assembly with flow prevention between the two prior touse. For instance, a device may include a release member positionedadjacent to both a reservoir and a microneedle array. The release membermay be separated from the device prior to use such that during use thereservoir and the microneedle array are in fluid communication with oneanother. Separation may be accomplished through the partial or completedetachment of the release member. For example, referring to FIGS. 12-17,one embodiment of a release member is shown that is configured to bedetached from a transdermal patch to initiate the flow of a drugcompound. More particularly, FIGS. 12-13 show a transdermal patch 300that contains a drug delivery assembly 370 and a microneedle assembly380. The drug delivery assembly 370 includes a reservoir 306 positionedadjacent to a rate control membrane 308.

The rate control membrane may help slow down the flow rate of the drugcompound upon its release. Specifically, fluidic drug compounds passingfrom the drug reservoir to the microneedle assembly via microfluidicchannels may experience a drop in pressure that results in a reductionin flow rate. If this difference is too great, some backpressure may becreated that may impede the flow of the compound and potentiallyovercome the capillary pressure of the fluid through the microfluidicchannels. Thus, the use of the rate control membrane may ameliorate thisdifference in pressure and allow the drug compound to be introduced intothe microneedle at a more controlled flow rate. The particularmaterials, thickness, etc. of the rate control membrane may vary basedon multiple factors, such as the viscosity of the drug compound, thedesired delivery time, etc.

The rate control membrane may be fabricated from permeable,semi-permeable or microporous materials that are known in the art tocontrol the rate of drug compounds and having permeability to thepermeation enhancer lower than that of drug reservoir. For example, thematerial used to form the rate control membrane may have an average poresize of from about 50 nanometers to about 5 micrometers, in someembodiments from about 100 nanometers to about 2 micrometers, and insome embodiments, from about 300 nanometers to about 1 micrometer (e.g.,about 600 nanometers). Suitable membrane materials include, forinstance, fibrous webs (e.g., woven or nonwoven), apertured films,foams, sponges, etc., which are formed from polymers such aspolyethylene, polypropylene, polyvinyl acetate, ethylene n-butyl acetateand ethylene vinyl acetate copolymers. Such membrane materials are alsodescribed in more detail in U.S. Pat. Nos. 3,797,494, 4,031,894,4,201,211, 4,379,454, 4,436,741, 4,588,580, 4,615,699, 4,661,105,4,681,584, 4,698,062, 4,725,272, 4,832,953, 4,908,027, 5,004,610,5,310,559, 5,342,623, 5,344,656, 5,364,630, and 6,375,978, which areincorporated in their entirety herein by reference for all relevantpurposes. A particularly suitable membrane material is available fromLohmann Therapie-Systeme.

Referring to FIGS. 12-13, although optional, the assembly 370 alsocontains an adhesive layer 304 that is positioned adjacent to thereservoir 306. The microneedle assembly 380 likewise includes a support312 from which extends a plurality of microneedles 330 having channels331, such as described above. The layers of the drug delivery assembly370 and/or the microneedle assembly 380 may be attached together ifdesired using any known bonding technique, such as through adhesivebonding, thermal bonding, ultrasonic bonding, etc.

Regardless of the particular configuration employed, the patch 300 alsocontains a release member 310 that is positioned between the drugdelivery assembly 370 and the microneedle assembly 380. While therelease member 310 may optionally be bonded to the adjacent support 312and/or rate control membrane 308, it is typically desired that it isonly lightly bonded, if at all, so that the release member 310 may beeasily withdrawn from the patch 300. If desired, the release member 310may also contain a tab portion 371 (FIGS. 12-13) that extends at leastpartly beyond the perimeter of the patch 300 to facilitate the abilityof a user to grab onto the member and pull it in the desired direction.In its “inactive” configuration as shown in FIGS. 12-13, the drugdelivery assembly 370 of the patch 300 securely retains a drug compound307 so that it does not flow to any significant extent into themicroneedles 330. The patch may be “activated” by simply applying aforce to the release member so that it is detached from the patch.

Referring to FIGS. 14-15, one embodiment for activating the patch 300 isshown in which the release member 310 is pulled in a longitudinaldirection. The entire release member 310 may be removed as shown inFIGS. 16-17, or it may simply be partially detached as shown in FIGS.14-15. In either case, however, the seal previously formed between therelease member 310 and the aperture (not shown) of the support 312 isbroken. In this manner, a drug compound 107 may begin to flow from thedrug delivery assembly 170 and into the channels 131 of the microneedles130 via the support 112. An exemplary illustration of how the drugcompound 307 flows from the reservoir 306 and into the channels 331 isshown in FIGS. 16-17. Notably, the flow of the drug compound 307 ispassively initiated and does not require any active displacementmechanisms (e.g., pumps).

In the embodiments shown in FIGS. 12-17, the detachment of the releasemember immediately initiates the flow of the drug compound to themicroneedles because the drug delivery assembly is already disposed influid communication with the microneedle assembly. In certainembodiments, however, it may be desired to provide the user with agreater degree of control over the timing of the release of the drugcompound. This may be accomplished by using a patch configuration inwhich the microneedle assembly is not initially in fluid communicationwith the drug delivery assembly. When it is desired to use the patch,the user may physically manipulate the two separate assemblies intofluid communication. The release member may be separated either beforeor after such physical manipulation occurs.

Referring to FIGS. 18-23, for example, one particular embodiment of apatch 200 is shown. FIGS. 18-19 illustrate the patch 200 before use, andshows a first section 250 formed by a microneedle assembly 280 and asecond section 260 formed by a drug delivery assembly 270. The drugdelivery assembly 270 includes a reservoir 206 positioned adjacent to arate control membrane 208 as described above. Although optional, theassembly 270 also contains an adhesive layer 204 that is positionedadjacent to the reservoir 206. The microneedle assembly 280 likewiseincludes a support 212 from which extends a plurality of microneedles230 having channels 231, such as described above.

In this embodiment, the support 212 and the rate control membrane 208are initially positioned horizontally adjacent to each other, and arelease member 210 extends over the support 212 and the rate controlmember 208. In this particular embodiment, it is generally desired thatthe release member 210 is releasably attached to the support 212 and therate control membrane 208 with an adhesive (e.g., pressure-sensitiveadhesive). In its “inactive” configuration as shown in FIGS. 18-19, thedrug delivery assembly 270 of the patch 200 securely retains a drugcompound 207 so that it does not flow to any significant extent into themicroneedles 230. When it is desired to “activate” the patch, therelease member 210 may be peeled away and removed, such as illustratedin FIGS. 20-21, to break the seal previously formed between the releasemember 210 and the aperture (not shown) of the support 212. Thereafter,the second section 260 may be folded about a fold line “F” as shown bythe directional arrow in FIG. 22 so that the rate control member 208 ispositioned vertically adjacent to the support 212 and in fluidcommunication therewith. Alternatively, the first section 250 may befolded. Regardless, folding of the sections 250 and/or 260 initiates theflow of a drug compound 207 from the drug delivery assembly 270 and intothe channels 231 of the microneedles 230 via the support 212 (See FIG.23).

The device may deliver an agent at a rate so as to be therapeuticallyuseful. In accord with this goal, the transdermal device may include ahousing with microelectronics and other micro-machined structures tocontrol the rate of delivery either according to a preprogrammedschedule or through active interface with the patient, a healthcareprofessional, or a biosensor. The device may include a material having apredetermined degradation rate, so as to control release of an siRNAconstruct contained within the device. The delivery rate may becontrolled by manipulating a variety of factors, including thecharacteristics of the formulation to be delivered (e.g., viscosity,electric charge, and/or chemical composition); the dimensions of thedevice (e.g., outer diameter and the volume of any openings); the numberof microneedles on a transdermal patch; the number of individual devicesin a carrier matrix; the application of a driving force (e.g., aconcentration gradient, a voltage gradient, a pressure gradient); theuse of a valve; and so forth.

Transportation of agents through the device may be controlled ormonitored using, for example, various combinations of valves, pumps,sensors, actuators, and microprocessors. These components may beproduced using standard manufacturing or microfabrication techniques.Actuators that may be useful with a device may include micropumps,microvalves, and positioners. For instance, a microprocessor may beprogrammed to control a pump or valve, thereby controlling the rate ofdelivery.

Flow of an agent through the device may occur based on diffusion orcapillary action, or may be induced using conventional mechanical pumpsor nonmechanical driving forces, such as electroosmosis orelectrophoresis, or convection. For example, in electroosmosis,electrodes are positioned on a biological surface (e.g., the skinsurface), a microneedle, and/or a substrate adjacent a microneedle, tocreate a convective flow which carries oppositely charged ionic speciesand/or neutral molecules toward or into the delivery site.

Flow of an agent may be manipulated by selection of the material formingthe microneedle surface. For example, one or more large grooves adjacentthe microneedle surface of the device may be used to direct the passageof the siRNA construct. Alternatively, the materials forming thenanostructured surface may be manipulated to either promote or inhibittransport of material along the surface, such as by controllinghydrophilicity or hydrophobicity.

The flow of an agent may be regulated using valves or gates as is knownin the art. Valves may be repeatedly opened and closed, or they may besingle-use valves. For example, a breakable barrier or one-way gate maybe installed in the device between a reservoir and the patternedsurface. When ready to use, the barrier may be broken or gate opened topermit flow through to the microneedle surface. Other valves or gatesused in the device may be activated thermally, electrochemically,mechanically, or magnetically to selectively initiate, modulate, or stopthe flow of molecules through the device. In one embodiment, flow iscontrolled by using a rate-limiting membrane as a “valve.”

In general, any agent delivery control system, including reservoirs,flow control systems, sensing systems, and so forth as are known in theart may be incorporated with devices. By way of example, U.S. Pat. Nos.7,250,037, 7,315,758, 7,429,258, 7,582,069, and 7,611,481 describereservoir and control systems as may be incorporated in devices.

During use, the presence of the nanostructured surface of themicroneedles within the skin may affect formation and maintenance ofcell/cell junctions including tight junctions and desmosomes. Aspreviously mentioned, tight junctions have been found in the stratumgranulosum and opening of the tight junctions may provide a paracellularroute for improved delivery of siRNA constructs.

The present disclosure may be further understood with reference to theExamples provided below.

EXAMPLE 1

Several different molds were prepared using photolithography techniquessimilar to those employed in the design and manufacture of electricalcircuits. Individual process steps are generally known in the art andhave been described

Initially, silicon substrates were prepared by cleaning with acetone,methanol, and isopropyl alcohol, and then coated with a 258 nanometer(nm) layer of silicon dioxide according to a chemical vapor depositionprocess.

A pattern was then formed on each substrate via an electron beamlithography patterning process as is known in the art using a JEOLJBX-9300FS EBL system. The processing conditions were as follows:

-   -   Beam current=11 nA    -   Acceleration voltage=100 kV    -   Shot pitch=14 nm    -   Dose=260 μC/cm²    -   Resist=ZEP520A, ˜330 nm thickness    -   Developer=n-amyl acetate    -   Development=2 min. immersion, followed by 30 sec. isopropyl        alcohol rinse.

A silicon dioxide etch was then carried out with an STS Advanced OxideEtch (AOE). Etch time was 50 seconds utilizing 55 standard cubiccentimeters per minute (sccm) He, 22 sccm CF₄, 20 sccm C₄F₈ at 4 mTorr,400 W coil, 200 W RIE and a DC Bias of 404-411 V.

Following, a silicon etch was carried out with an STS silicon oxide etch(SOE). Etch time was 2 minutes utilizing 20 sccm Cl₂ and 5 sccm Ar at 5mTorr, 600 W coil, 50 W RIE and a DC Bias of 96-102 V. The silicon etchdepth was 500 nm.

A buffered oxide etchant (BOE) was used for remaining oxide removal thatincluded a three minute BOE immersion followed by a deionized waterrinse.

An Obducat NIL-Eitre® 6 nanoimprinter was used to form nanopatterns on avariety of polymer substrates. External water was used as coolant. TheUV module utilized a single pulsed lamp at a wave length of between 200and 1000 nm at 1.8 W/cm². A UV filter of 250-400 nm was used. Theexposure area was 6 inches with a maximum temperature of 200° C. and 80Bar. The nanoimprinter included a semi-automatic separation unit andautomatic controlled demolding.

To facilitate the release of the nanoimprinted films from the molds, themolds were treated with Trideca-(1,1,2,2-tetrahydro)-octytrichlorosilane(F₁₃-TCS). To treat a mold, the silicon mold was first cleaned with awash of acetone, methanol, and isopropyl alcohol and dried with anitrogen gas. A Petri dish was placed on a hot plate in a nitrogenatmosphere and 1-5 ml of the F₁₃-TCS was added to the Petri dish. Asilicon mold was placed in the Petri dish and covered for 10-15 minutesto allow the F₁₃-TCS vapor to wet out the silicon mold prior to removalof the mold.

Five different polymers as given in Table 1, below, were utilized toform various nanotopography designs.

TABLE 1 Glass Surface Transition Tensile Tension Temperature, Modulus(mN/m) Polymer T_(g) (K) (MPa) @20° C. Polyethylene 140-170 100-300 30Polypropylene 280 1,389 21 PMMA 322 3,100 41 Polystyrene 373 3,300 40Polycarbonate 423 2,340 43

Several different nanotopography patterns were formed, schematicrepresentations of which are illustrated in FIGS. 24A-24D. Thenanotopography pattern illustrated in FIG. 24E was a surface of a flatsubstrate purchased from NTT Advanced Technology of Tokyo, Japan. Thepatterns were designated DN1 (FIG. 24A), DN2 (FIG. 24B), DN3 (FIG. 24C),DN4 (FIG. 24D) and NTTAT2 (FIG. 24E). SEM images of the molds are shownin FIGS. 24A, 24B, and 24C, and images of the films are shown in FIGS.24D and 24E. FIG. 8 illustrates a nanopatterned film formed by use ofthe mold of FIG. 24A (DN1). In this particular film, the polymerfeatures were drawn by temperature variation as previously discussed.The surface roughness of the pattern of FIG. 24E was found to be 34 nm.

The pattern illustrated in FIGS. 7C and 7D was also formed according tothis nanoimprinting process. This pattern included the pillars 72 andpillars 62, as illustrated. Larger pillars 72 were formed with a 3.5micrometer (μm) diameter and 30 μm heights with center-to-center spacingof 6.8 μm. Pillars 62 were 500 nm in height and 200 nm in diameter and acenter-to-center spacing of 250 nm.

The nanoimprinting process conditions used with polypropylene films areprovided below in Table 2.

TABLE 2 Pressure Time (s) Temperature (C.) (Bar) 10 50 10 10 75 20 10100 30 420 160 40 180 100 40 180 50 40 180 25 40

EXAMPLE 2

Films were formed as described above in Example 1 including variousdifferent patterns and formed of either polystyrene (PS) orpolypropylene (PP). The underlying substrate varied in thickness.Patterns utilized were either DN2, DN3, or DN4 formed utilizingformation processes as described in Example 1. The pattern molds werevaried with regard to hole depth and feature spacing to form a varietyof differently-sized features having the designated patterns. Sample no.8 (designated BB1) was formed by use of a 0.6 μm millipore polycarbonatefilter as a mold. A 25 μm polypropylene film was laid over the top ofthe filter and was then heated to melt such that the polypropylene couldflow into the pores of the filter. The mold was then cooled and thepolycarbonate mold dissolved by use of a methylene chloride solvent.

SEMs of the formed films are illustrated in FIGS. 25-33 andcharacteristics of the formed films are summarized in Table 3, below.

TABLE 3 Film Cross Surface Water Sample thickness Pattern SectionalFeature Aspect Roughness Fractal Contact No. Fig. Pattern Material (μm)Feature¹ Dimension² height³ Ratio (nm) Dimension Angle 1 25 DN3 PS 75 A1100 nm  520 nm 0.47 150 2.0 100° B 400 nm 560 nm 1.4 C 200 nm 680 nm3.4 2 26A, 26B DN2 PP 5.0 n/a 200 nm 100 nm 0.5 16 2.15  91° 3 27 DN2 PS75 n/a 200 nm 1.0 μm 5 64 2.2 110° 4 28 DN2 PP 25.4 n/a 200 nm 300 nm1.5 38 1.94 118° 5 29 DN3 PS 75 A 1100 nm  570 nm 0.52 21.1 1.98 100° B400 nm 635 nm 1.6 C 200 nm — — 6 30 DN4 PS 75 n/a 200 nm — — 30.6 2.04 80° 7 31 DN4 PP 25.4 n/a 200 nm — — 21.4 2.07 112° 8 32 BB1 PP 25.4 n/a600 nm 18 μm 30 820 2.17 110° 9 33 DN3 PP 5 A 1100 nm  165 nm 0.15 502.13 — B 400 nm 80 nm 0.2 C 200 nm 34 nm 0.17 ¹Pattern Features as shownon the figures. ²Cross sectional dimension values were derived from themold and equated as an approximation of the maximum dimension of thestructure, although it should be understood that the actual dimension ofany given individual structure may vary slightly as may be seen in thefigures. ³Feature heights are provided as the average of severalindividually determined feature heights.

For each sample AFM was utilized to characterize the film.Characterizations included formation of scanning electron micrograph(SEM), determination of surface roughness, determination of maximummeasured feature height, and determination of fractal dimension.

The atomic force microscopy (AFM) probe utilized was a series 16 siliconprobe and cantilever available from μMasch. The cantilever had aresonant frequency of 170 kHz, a spring constant of 40 N/m, a length of230±5 μm, a width of 40±3 μm, and a thickness of 7.0±0.5 μm. The probetip was an n-type phosphorous-doped silicon probe, with a typical probetip radius of 10 nm, a full tip cone angle of 40°, a total tip height of20-25 μm, and a bulk resistivity 0.01-0.05 ohm-cm.

The surface roughness value given in Table 3 is the arithmetical meanheight of the surface areal roughness parameter as defined in the ISO25178 series.

The Fractal Dimension was calculated for the different angles byanalyzing the Fourier amplitude spectrum; for different angles theamplitude Fourier profile was extracted and the logarithm of thefrequency and amplitude coordinates calculated. The fractal dimension,D, for each direction is then calculated asD=(6+s)/2,

where s is the (negative) slope of the log-log curves. The reportedfractal dimension is the average for all directions.

The fractal dimension may also be evaluated from 2D Fourier spectra byapplication of the Log Log function. If the surface is fractal the LogLog graph should be highly linear, with at negative slope (see, e.g.,Fractal Surfaces, John C. Russ, Springer-Verlag New York, LLC, July,2008).

EXAMPLE 3

HaCaT human skin epithelial cells were grown in DMEM, 10% FBS, 1%penicillin/streptomycin at 37° C., 5% CO₂ for 24 hours at aconcentration of 25,000 cell/cm² in 6 well plates. Plates either hadpolypropylene nanopatterned films formed as described above in Example 1and designate DN1, DN2 (Sample 4 of Table 3), DN3 or untreated surfaceat the bottom of the well. Nanopatterned films were adhered in placewith cyanoacrylate.

Cells were detached from the surfaces with 1 mL of trypsin per well for10 minutes, quenched with 1 mL growth medium (same as above), thentransferred to a microfuge tube and pelleted at 1200 rpm for 7 minutes.

RNA was isolated from pelleted cells using the RNeasy miniprep kit fromQiagen using the manufacturer's protocol. Briefly, cells were lysed,mixed with ethanol and spun down in a column. Lysates were then washed 3times, treated with DNase and eluted in 40 μl volumes.

cDNA was created from the RNA isolated using the RT first strand kitfrom SA Biosciences. Briefly, RNA was treated with DNase again at 42° C.for 5 minutes. Random primers and reverse transcriptase enzyme was thenadded and incubated at 42° C. for 15 minutes, then incubated at 95° C.for 5 minutes to stop reaction.

qPCR was then performed on the cDNA samples using RT profiler custom PCRarray from SA Biosciences with primers for IL1-β, IL6, IL8, IL10, IL1R1,TNFα, TGFβ-1, PDGFA, GAPDH, HDGC, RTC and PPC. Briefly, cDNA was mixedwith SYBR green and water, and then added to a PCR plate pre-fixed withthe correct sense and antisense primer pair for the gene of interest.The plate was then run on an ABI StepOnePlus PCR machine heated to 95°C. for 10 minutes, then for 45 cycles of: 15 seconds at 95° C. and 1minute at 60° C.

Delta delta C_(T) analysis was performed using GAPDH as the internalcontrol. HDGC, RTC and PPC levels were used as additional internalcontrols for activity and genomic DNA contamination.

One-way ANOVA and Tukey's 2-point tests were then used to determinestatistical significance in the differences between surfaces.

Table 4, below, presents the protein expressions obtained as the foldchange in expression on nanoimprinted structures produced onpolypropylene films versus expression on an unstructured film.

TABLE 4 Mold IL1-β IL6 IL8 IL10 IL1R1 TNFα TGFβ1 PDGFA DN1 2.24 3.330.36 1.17 0.6 0.57 0.37 1.37 DN2 3.18 3.2 0.46 0.43 0.36 0.57 0.42 1.23DN3 3.36 2.7 0.47 5.83 1.6 0.37 0.35 0.64

EXAMPLE 4

HaCaT human skin epithelial cells were grown in DMEM, 10% FBS, 1%penicillin/streptomycin at 37° C., 5% CO₂ for 24 hours at aconcentration of 25,000 cell/cm² in 6 well plates. Plates either had apolypropylene film formed as described above in Example 1 withdesignation DN1, DN2 (Sample 4 of Table 3), DN3 or an untreated surfaceat the bottom of the well. Films were adhered in place withcyanoacrylate.

Media was collected from each well and analyzed for cytokine productionwith a Milliplex Map Kit from Millipore. Beads to detect IL1-β, IL1RA,IL6, IL8, IL10, PDGF-AA, PGGF-AB/BB and TNF-α were used. Readings weredone on a BioRad BioPlex machine. Briefly, media was placed intomicroplate wells with filters. Primary beads were added and incubated atroom temperature for 1 hour with shaking. The plates were then washedand incubated with detection antibodies for 30 minutes at roomtemperature with shaking. Strepavidin-phycoertythrin was then added andincubated at room temperature for an additional 30 minutes. Plates werethen washed, beads were resuspended in assay buffer and medianfluorescent intensity was analyzed on the BioPlex.

EXAMPLE 5

The permeability effects of films patterned as described herein weredetermined on a monolayer of Caco-2 cells (human epithelial colorectaladenocarcinoma cells).

Films formed as described above in Example 1 were utilized includingfilms formed with patterns designated as DN2, DN3, and DN4. A fourthfilm, designated as BB1 (described in Example 2, above) was also used.The protocol was run with multiple examples of each film type.

The general protocol followed for each film was as follows:

Materials

Cell culture inserts 0.4 um pore size HDPET membrane (BD Falcon)

24 well plate (BD Falcon)

Caco-2 media

Nanostructured membranes as described above

IgG-FITC (Sigma Aldrich)

BSA-FITC (Sigma Aldrich)

Minimum Essential Medium no phenol red (Invitrogen)

TEER voltmeter

Warmed PBS

Black 96-well plate

Aluminum foil

Protocol

-   -   1. Seed Caco-2 cells on collagen coated well inserts 2 weeks        before permeability assay is to be performed. Collagen coated        plates are made by making a 1:1 volume of 100% ethanol to        collagen. Dry surfaces in sterile hood overnight until dry.    -   2. Make 0.1 mg/mL solution of FITC-conjugated molecule (BSA,        IgG, etc) of interest in phenol red free Alpha MEM media. Wrap        in aluminum foil to protect from light.    -   3. Check for confluency of Caco-2 cells by measuring the        resistance. Resistance should be above ˜600 Ohms for confluency.    -   4. Aspirate old media from cell culture inserts on apical and        basolateral sides. Rinse with PBS to remove any residual        phenol-red dye.    -   5. Add 0.5 mL of FITC-conjugated solution on apical side of each        insert.    -   6. In another 24 well plate with cell culture inserts, add 0.5        mL of warmed PBS.    -   7. Transfer inserts to the plate with PBS. Blot the bottom of        the insert on a Kim wipe to remove residual phenol red.    -   8. t=0—time point: sample 75 μL from the basolateral side of        insert and transfer to a black-bottom 96-well plate. Replace the        volume with 75 μL of warmed PBS. Record the resistance of each        well using the “chopstick” electrodes.    -   9. Carefully add the membrane to the appropriately labeled well.        Controls are the unimprinted membranes and the cells alone.        Check under a microscope that the membranes make direct contact        to the cells. You should be able to see a sharp circle,        indicating contact with the cells.    -   10. t=0 time point: repeat step 7 and then place in the        incubator for 1 hour    -   11. t=1 time point: repeat step 7 and then place in the        incubator for 1 hour    -   12. t=2 time point: repeat step 7    -   13. Measure fluorescence signal using a spectrofluorometer plate        reader. FITC (excitation=490 nm, emission=520 nm)        Results

Films utilized and results obtained are summarized in Table 5, below.

TABLE 5 Sample no. (see Table 3) 2 3 4 5 6 7 8 Pattern DN2 DN2 DN2 DN3DN4 DN4 BB1 Material PP PS PP PS PS PP PP Effective 5.3 16.3 0.29 10.432.3 4.8 7.8 Compression Modulus (MPa) Effective Shear 5.32 58.9 218 31977.8 4.4 26.7 Modulus (MPa) BET Surface — 0.11 — 0.44 — 4.15 — Area(m²/g) BSA permeability — 2 1.9 3.3 2 1.4 1 increase at 120 min. (MW 66kDa) IgG permeability — 1 — 1 3.5 — — increase at 120 min. (MW 150 kDa)

Moduli were determined according to standard methods as are known in theart as described by Schubert, et al. (Sliding induced adhesion of stiffpolymer microfiber arrays: 2. Microscale behaviour, Journal RoyalSociety, Interface, Jan. 22, 2008. 10.1098/rsif.2007.1309)

The contact angles were measured by placing a drop of water on thesurface according to standard practice. (See, e.g., Woodward, First TenAngstroms, Portsmouth, Va.).

FIG. 34 graphically illustrates the effects on permeability to bovineserum albumin (BSA) in a monolayer of cells on polystyrene filmspatterned with nanopatterns as described herein. The film patternsincluded a DN2 pattern (sample no. 3), a DN3 pattern (sample no. 5), anda DN4 pattern (sample no. 6), as indicated. Also shown are results for anon-patterned film (marked PSUI on FIG. 34) and a layer of cells with noadjacent film (marked ‘cells’ on FIG. 22).

FIG. 35 graphically illustrates the effects on permeability toimmunoglobulin-G (IgG) in a monolayer of cells on polystyrene filmspatterned with nanopatterns as described herein. The film patternsincluded a DN2 pattern (sample no. 3), a DN3 pattern (sample no. 5), anda DN4 pattern (sample no. 6), as indicated. Also shown are results for anon-patterned film (marked PSUI on FIG. 35) and a layer of cells with noadjacent film (marked ‘cells’ on FIG. 35).

The BSA signal was read on a fluorometer and the IgG signal was read ona spectrophotometer.

FIGS. 36A and 36B are 3D live/dead flourescein staining images showingparacellular transport of IgG across a monolayer of cells on apolystyrene DN4 patterned surface (sample no. 6).

FIG. 37 graphically illustrates the effects on permeability to BSA in amonolayer of cells on polypropylene films patterned with nanopatterns asdescribed herein. Patterns included BB1 (sample no. 8), DN2 (sample no.4), and DN4 (sample no. 7), as indicated. Also shown are results for anon-patterned film (marked PSUI on FIG. 37) and a layer of cells with noadjacent film (marked ‘cells’ on FIG. 37).

FIG. 38 graphically illustrates the effects on permeability to IgG in amonolayer of cells on polypropylene films patterned with nanopatterns asdescribed herein. Patterns included BB1 (sample no. 8), DN2 (sample no.4), and DN4 (sample no. 7), as indicated. Also shown are results for anon-patterned film (marked PSUI on FIG. 38) and a layer of cells with noadjacent film (marked ‘cells’ on FIG. 38).

FIGS. 39A and 39B are 3D live/dead flourescein staining images showingparacellular transport of IgG across a monolayer of cells on apolypropylene DN2 patterned surface (sample no. 4).

FIGS. 40A-40F are scanning electron microscopy (SEM) images of Caco-2cells cultured on nanopatterned surfaces. Specifically, FIGS. 40A and40B illustrate Caco-2 cells on a flat polystyrene control film. FIGS.40C and 40D illustrate Caco-2 cells on a polystyrene film patterned witha DN2 pattern (sample no. 3) as described above, and FIGS. 40E and 40Fillustrate Caco-2 cells on a polystyrene film patterned with a DN3pattern (sample no. 5) as described above.

EXAMPLE 6

An array of microneedles including a nanopatterned surface was formed.

Initially, an array of microneedles as illustrated in FIG. 2 was formedon a silicon wafer via a photolithography process. Each needle includedtwo oppositely placed side channels, aligned with one through-die holein the base of the needle (not visible on FIG. 2).

Microneedles were formed according to a typical micromachining processon a silicon based wafer. The wafers were layered with resist and/oroxide layers followed by selective etching (oxide etching, DRIE etching,iso etching), resist stripping, oxide stripping, and lithographytechniques (e.g., iso lithography, hole lithography, slit lithography)according to standard methods to form the array of microneedles.

Following formation of the microneedle array, a 5 μm polypropylene filmincluding a DN2 pattern formed thereon as described above in Example 1,the characteristics of which are described at sample 2 in Table 3, waslaid over the microneedle array. The wafer/film structure was held on aheated vacuum box (3 in. H₂O vacuum) at elevated temperature (130° C.)for a period of one hour to gently pull the film over the surface of themicroneedles while maintaining the nanopatterned surface of the film.

FIG. 41 illustrates the film over the top of the array of microneedles,and FIG. 42 is a closer view of a single needle of the array includingthe nanopatterned film overlaying the top of the needle.

EXAMPLE 7

Methods as described in Example 5 were utilized to determine thepermeability effects of films patterned as described herein on amonolayer of Caco-2 cells when considering permeability of a cell layerto siRNA.

siRNA utilized was the BLOCK-iT™ Fluorescent Oligo available fromInvitrogen. The siRNA is a fluorescein-labeled dsRNA oligomer. Theprotocol was the same as described in Example 5. The structured filmused included a DN2 pattern on a polypropylene film (sample 4 of Table3) as well as an unpatterned film (PPUI) and a layer of cells with nofilm (cells). Results of permeability over time are shown in FIG. 43.

While the subject matter has been described in detail with respect tothe specific embodiments thereof, it will be appreciated that thoseskilled in the art, upon attaining an understanding of the foregoing,may readily conceive of alterations to, variations of, and equivalentsto these embodiments. Accordingly, the scope of the present disclosureshould be assessed as that of the appended claims and any equivalentsthereto.

What is claimed is:
 1. A device for delivery of an siRNA constructacross a dermal barrier, the device comprising: a microneedle and aplurality of nanostructures and microstructures projecting directlyoutward from a surface of the microneedle in a predetermined pattern,wherein at least a portion of the microstructures have a cross-sectionaldimension of greater than about 500 nanometers and less than about 10micrometers and a height of from about 20 nanometers to about 1micrometer, and wherein at least a portion of the nanostructures have across-sectional dimension of less than about 500 nanometers and greaterthan about 5 nanometers and an aspect ratio of from about 0.2 to about5, wherein the microneedle further contains a channel; and a reservoirthat is in fluid communication with the channel of the microneedle andthat contains an siRNA construct.
 2. The device of claim 1, furthercomprising second nanostructures having a cross-sectional dimension lessthan the cross-sectional dimension of the microstructures and greaterthan the cross-sectional dimension of the first nanostructures.
 3. Thedevice of claim 1, wherein at least a portion of the nanostructures havea center-to-center spacing of from about 50 nanometers to about 1micrometer.
 4. The device of claim 1, wherein at least a portion of thenanostructures have a height of from about 10 nanometers to about 1micrometer.
 5. The device of claim 1, wherein at least a portion of thenanostructures have an aspect ratio of from about 0.5 to about 3.5. 6.The device of claim 1, wherein the siRNA agent includes a duplexedregion of between about 20 and about 30 pairs.
 7. The device of claim 1,at least one strand of the siRNA including a 3′ overhang of two or threenucleotides.
 8. The device of claim 1, the siRNA agent comprisingadditional nucleotides or nucleotide analogue.
 9. The device of claim 1,wherein the siRNA agent comprises a nucleotide sequence that isidentical to a portion of a targeted gene.
 10. The device of claim 1,wherein the siRNA agent includes insertions, deletions, or single pointmutations compared to a portion of a targeted gene.
 11. The device ofclaim 1, wherein the siRNA agent is a single strand siRNA.
 12. Thedevice of any of claim 1, wherein the siRNA comprises a ligand tetheredto the siRNA.
 13. The device of claim 1, wherein the siRNA agent isincorporated into a delivery vehicle.
 14. The device of claim 1, whereinthe siRNA construct comprises a vector.
 15. The device according toclaim 1, wherein the pattern has a fractal dimension of greater thanabout
 1. 16. The device according to claim 1, wherein at least a portionof the nanostructures have a cross-sectional dimension of from about 100to about 300 nanometers.
 17. The device according to claim 1, whereinthe nanostructures have approximately the same cross-sectionaldimension.
 18. The device according to claim 1, wherein the ratio of thecross sectional dimension of two adjacent nanostructures to thecenter-to-center spacing between those two structures is between about1:1 and about 1:4.
 19. The device according to claim 1, wherein at leasta portion of the nanostructures have an equidistant spacing.
 20. Thedevice according to claim 1, wherein at a portion of the nanostructuresare in the form of pillars.
 21. The device according to claim 1, whereinthe channel has a cross-sectional dimension of from about 1 to about 100micrometers.
 22. The device according to claim 21, wherein the channelhas a length of from about 10 to about 800 micrometers.
 23. The deviceaccording to claim 1, wherein the device contains a microneedle arraythat contains the microneedle.
 24. The device according to claim 23,wherein the array contains a base having an aperture, wherein theaperture is in at least partial alignment with the channel of themicroneedle.
 25. The device according to claim 24, wherein the reservoiris attached to a substrate upon which the array is located.
 26. Thedevice according to claim 24, wherein the reservoir is removablyconnected to the array.
 27. The device according to claim 1, wherein atleast a portion of the nanostructures have a cross-sectional dimensionof from about 20 to about 400 nanometers and at least a portion of themicrostructures have a cross-sectional dimension of from about 600nanometers to about 1.5 micrometers.
 28. The device according to claim1, wherein at least a portion of the microstructures have an aspectratio of from about 0.2 to about
 5. 29. The device according to claim 1,wherein the cross-sectional dimension of the microstructures is greaterthan the height of the microstructures.
 30. The device according toclaim 29, wherein at least a portion of the microstructures have anaspect ratio of from about 0.15 to
 1. 31. The device according to claim1, wherein at least a portion of the nanostructures have a heightgreater than a cross-sectional dimension.
 32. A method for delivering ansiRNA construct across a dermal barrier, the method comprising:penetrating a stratum corneum with a microneedle having a surface onwhich a plurality of nanostructures and microstructures project directlyoutward therefrom in a predetermined pattern, wherein the microneedlefurther contains a channel, wherein at least a portion of themicrostructures nave a cross-sectional dimension of greater than about500 nanometers and less than about 10 micrometers and a height of fromabout 20 nanometers to about 1 micrometer, and wherein at least aportion of the nanostructures have a cross-section dimension of lessthan about 500nanometers and greater than about 5 nanometers and anaspect ratio of from about 0.2 to about 5, transporting an siRNAconstruct from a reservoir through the channel of the microneedle andacross the stratum corneum.